US20020061564A1 - Method for chemical transformation using a mutated enzyme - Google Patents
Method for chemical transformation using a mutated enzyme Download PDFInfo
- Publication number
- US20020061564A1 US20020061564A1 US10/039,952 US3995201A US2002061564A1 US 20020061564 A1 US20020061564 A1 US 20020061564A1 US 3995201 A US3995201 A US 3995201A US 2002061564 A1 US2002061564 A1 US 2002061564A1
- Authority
- US
- United States
- Prior art keywords
- enzyme
- target
- mutated
- catalyzes
- mutated enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 171
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 171
- 238000000034 method Methods 0.000 title claims abstract description 118
- 238000006243 chemical reaction Methods 0.000 title description 43
- 150000002576 ketones Chemical class 0.000 claims abstract description 59
- 150000001413 amino acids Chemical class 0.000 claims abstract description 37
- 238000006722 reduction reaction Methods 0.000 claims abstract description 36
- 150000001412 amines Chemical class 0.000 claims abstract description 35
- 238000006268 reductive amination reaction Methods 0.000 claims abstract description 33
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 29
- 239000011541 reaction mixture Substances 0.000 claims abstract description 29
- 238000004519 manufacturing process Methods 0.000 claims abstract description 28
- 230000009467 reduction Effects 0.000 claims abstract description 23
- 238000005891 transamination reaction Methods 0.000 claims abstract description 17
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 8
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims description 48
- 102000007698 Alcohol dehydrogenase Human genes 0.000 claims description 39
- 108010021809 Alcohol dehydrogenase Proteins 0.000 claims description 39
- 239000000203 mixture Substances 0.000 claims description 36
- 101710088194 Dehydrogenase Proteins 0.000 claims description 29
- 235000005152 nicotinamide Nutrition 0.000 claims description 25
- 229960003966 nicotinamide Drugs 0.000 claims description 24
- 239000011570 nicotinamide Substances 0.000 claims description 24
- 230000008859 change Effects 0.000 claims description 23
- 108010028658 Leucine Dehydrogenase Proteins 0.000 claims description 22
- 239000007793 ph indicator Substances 0.000 claims description 18
- 230000000694 effects Effects 0.000 claims description 17
- 102000005751 Alcohol Oxidoreductases Human genes 0.000 claims description 8
- 108010031132 Alcohol Oxidoreductases Proteins 0.000 claims description 8
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 8
- IAWVHZJZHDSEOC-UHFFFAOYSA-N 3,3-dimethyl-2-oxobutanoic acid Chemical compound CC(C)(C)C(=O)C(O)=O IAWVHZJZHDSEOC-UHFFFAOYSA-N 0.000 claims description 7
- 108090000340 Transaminases Proteins 0.000 claims description 5
- 102000003929 Transaminases Human genes 0.000 claims description 5
- 101001110310 Lentilactobacillus kefiri NADP-dependent (R)-specific alcohol dehydrogenase Proteins 0.000 claims description 4
- 229910021529 ammonia Inorganic materials 0.000 claims description 4
- MSCJSMVNUFGBHB-UHFFFAOYSA-N 3-naphthalen-1-yl-2-oxopropanoic acid Chemical compound C1=CC=C2C(CC(=O)C(=O)O)=CC=CC2=C1 MSCJSMVNUFGBHB-UHFFFAOYSA-N 0.000 claims description 2
- GTZXBCWKXWCNNO-UHFFFAOYSA-N 3-naphthalen-2-yl-2-oxopropanoic acid Chemical compound C1=CC=CC2=CC(CC(=O)C(=O)O)=CC=C21 GTZXBCWKXWCNNO-UHFFFAOYSA-N 0.000 claims description 2
- FKWWGKVXQZUMFW-UHFFFAOYSA-N CP(=O)CCC(=O)C(O)=O Chemical compound CP(=O)CCC(=O)C(O)=O FKWWGKVXQZUMFW-UHFFFAOYSA-N 0.000 claims description 2
- 101100533888 Hypocrea jecorina (strain QM6a) sor4 gene Proteins 0.000 claims description 2
- 101100053441 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) YPR1 gene Proteins 0.000 claims description 2
- 230000003287 optical effect Effects 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 108030000991 Aromatic-amino-acid transaminases Proteins 0.000 claims 2
- 108010088278 Branched-chain-amino-acid transaminase Proteins 0.000 claims 2
- 108010078226 phenylalanine oxidase Proteins 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 abstract description 39
- 230000001131 transforming effect Effects 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 48
- KWOLFJPFCHCOCG-UHFFFAOYSA-N Acetophenone Chemical compound CC(=O)C1=CC=CC=C1 KWOLFJPFCHCOCG-UHFFFAOYSA-N 0.000 description 38
- 238000001514 detection method Methods 0.000 description 35
- 229940024606 amino acid Drugs 0.000 description 31
- 235000001014 amino acid Nutrition 0.000 description 30
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 27
- 238000007254 oxidation reaction Methods 0.000 description 26
- 102000004316 Oxidoreductases Human genes 0.000 description 22
- 108090000854 Oxidoreductases Proteins 0.000 description 22
- 239000000047 product Substances 0.000 description 22
- -1 gamma-amino acids Chemical class 0.000 description 19
- 239000000872 buffer Substances 0.000 description 17
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 15
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 14
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 14
- 229960000723 ampicillin Drugs 0.000 description 14
- 239000008188 pellet Substances 0.000 description 14
- 230000003647 oxidation Effects 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- 238000012216 screening Methods 0.000 description 13
- 239000003153 chemical reaction reagent Substances 0.000 description 12
- 238000002703 mutagenesis Methods 0.000 description 11
- 231100000350 mutagenesis Toxicity 0.000 description 11
- 230000002829 reductive effect Effects 0.000 description 11
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 10
- 239000000725 suspension Substances 0.000 description 10
- 239000008057 potassium phosphate buffer Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- JYAQYXOVOHJRCS-UHFFFAOYSA-N 1-(3-bromophenyl)ethanone Chemical compound CC(=O)C1=CC=CC(Br)=C1 JYAQYXOVOHJRCS-UHFFFAOYSA-N 0.000 description 8
- BUZYGTVTZYSBCU-UHFFFAOYSA-N 1-(4-chlorophenyl)ethanone Chemical compound CC(=O)C1=CC=C(Cl)C=C1 BUZYGTVTZYSBCU-UHFFFAOYSA-N 0.000 description 8
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- ZPLCXHWYPWVJDL-UHFFFAOYSA-N 4-[(4-hydroxyphenyl)methyl]-1,3-oxazolidin-2-one Chemical compound C1=CC(O)=CC=C1CC1NC(=O)OC1 ZPLCXHWYPWVJDL-UHFFFAOYSA-N 0.000 description 7
- 101000798396 Bacillus licheniformis Phenylalanine racemase [ATP hydrolyzing] Proteins 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 7
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 7
- 239000006142 Luria-Bertani Agar Substances 0.000 description 7
- 108010006785 Taq Polymerase Proteins 0.000 description 7
- 150000001298 alcohols Chemical class 0.000 description 7
- 150000001299 aldehydes Chemical class 0.000 description 7
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 7
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 7
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 7
- 230000002596 correlated effect Effects 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 238000007747 plating Methods 0.000 description 7
- 108091008146 restriction endonucleases Proteins 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 description 6
- OBRMNDMBJQTZHV-UHFFFAOYSA-N cresol red Chemical compound C1=C(O)C(C)=CC(C2(C3=CC=CC=C3S(=O)(=O)O2)C=2C=C(C)C(O)=CC=2)=C1 OBRMNDMBJQTZHV-UHFFFAOYSA-N 0.000 description 6
- 150000004715 keto acids Chemical class 0.000 description 6
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 6
- 238000006479 redox reaction Methods 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 6
- RQEUFEKYXDPUSK-SSDOTTSWSA-N (1R)-1-phenylethanamine Chemical compound C[C@@H](N)C1=CC=CC=C1 RQEUFEKYXDPUSK-SSDOTTSWSA-N 0.000 description 5
- 108020005199 Dehydrogenases Proteins 0.000 description 5
- GKKZMYDNDDMXSE-UHFFFAOYSA-N Ethyl 3-oxo-3-phenylpropanoate Chemical compound CCOC(=O)CC(=O)C1=CC=CC=C1 GKKZMYDNDDMXSE-UHFFFAOYSA-N 0.000 description 5
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 5
- 235000019270 ammonium chloride Nutrition 0.000 description 5
- 230000000875 corresponding effect Effects 0.000 description 5
- OKANYBNORCUPKZ-UHFFFAOYSA-N ethyl 2-ethyl-3-oxobutanoate Chemical compound CCOC(=O)C(CC)C(C)=O OKANYBNORCUPKZ-UHFFFAOYSA-N 0.000 description 5
- 0 *C(B)=O.*C([H])(B)O Chemical compound *C(B)=O.*C([H])(B)O 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000007795 chemical reaction product Substances 0.000 description 4
- 150000002019 disulfides Chemical class 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- OHLRLMWUFVDREV-UHFFFAOYSA-N ethyl 4-chloro-3-oxobutanoate Chemical compound CCOC(=O)CC(=O)CCl OHLRLMWUFVDREV-UHFFFAOYSA-N 0.000 description 4
- 239000000543 intermediate Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 4
- 239000008247 solid mixture Substances 0.000 description 4
- ULMJQMDYAOJNCC-ZCFIWIBFSA-N (1r)-1-(3-bromophenyl)ethanol Chemical compound C[C@@H](O)C1=CC=CC(Br)=C1 ULMJQMDYAOJNCC-ZCFIWIBFSA-N 0.000 description 3
- MVOSNPUNXINWAD-ZCFIWIBFSA-N (1r)-1-(4-chlorophenyl)ethanol Chemical compound C[C@@H](O)C1=CC=C(Cl)C=C1 MVOSNPUNXINWAD-ZCFIWIBFSA-N 0.000 description 3
- WAPNOHKVXSQRPX-SSDOTTSWSA-N (R)-1-phenylethanol Chemical compound C[C@@H](O)C1=CC=CC=C1 WAPNOHKVXSQRPX-SSDOTTSWSA-N 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 150000001728 carbonyl compounds Chemical class 0.000 description 3
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 3
- 239000013592 cell lysate Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- 239000002198 insoluble material Substances 0.000 description 3
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 3
- 229910000160 potassium phosphate Inorganic materials 0.000 description 3
- 235000011009 potassium phosphates Nutrition 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 150000003573 thiols Chemical class 0.000 description 3
- LIBZHYLTOAGURM-ZCFIWIBFSA-N (1r)-1-(3-bromophenyl)ethanamine Chemical compound C[C@@H](N)C1=CC=CC(Br)=C1 LIBZHYLTOAGURM-ZCFIWIBFSA-N 0.000 description 2
- PINPOEWMCLFRRB-ZCFIWIBFSA-N (1r)-1-(4-chlorophenyl)ethanamine Chemical compound C[C@@H](N)C1=CC=C(Cl)C=C1 PINPOEWMCLFRRB-ZCFIWIBFSA-N 0.000 description 2
- NPDBDJFLKKQMCM-BYPYZUCNSA-N (2r)-2-azaniumyl-3,3-dimethylbutanoate Chemical compound CC(C)(C)[C@@H]([NH3+])C([O-])=O NPDBDJFLKKQMCM-BYPYZUCNSA-N 0.000 description 2
- WAPNOHKVXSQRPX-ZETCQYMHSA-N (S)-1-phenylethanol Chemical compound C[C@H](O)C1=CC=CC=C1 WAPNOHKVXSQRPX-ZETCQYMHSA-N 0.000 description 2
- WAPNOHKVXSQRPX-UHFFFAOYSA-N 1-phenylethanol Chemical compound CC(O)C1=CC=CC=C1 WAPNOHKVXSQRPX-UHFFFAOYSA-N 0.000 description 2
- RQEUFEKYXDPUSK-UHFFFAOYSA-N 1-phenylethylamine Chemical compound CC(N)C1=CC=CC=C1 RQEUFEKYXDPUSK-UHFFFAOYSA-N 0.000 description 2
- OYCLSQDXZMROJK-UHFFFAOYSA-N 2-bromo-4-[3-(3-bromo-4-hydroxyphenyl)-1,1-dioxo-2,1$l^{6}-benzoxathiol-3-yl]phenol Chemical compound C1=C(Br)C(O)=CC=C1C1(C=2C=C(Br)C(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 OYCLSQDXZMROJK-UHFFFAOYSA-N 0.000 description 2
- OLQIKGSZDTXODA-UHFFFAOYSA-N 4-[3-(4-hydroxy-2-methylphenyl)-1,1-dioxo-2,1$l^{6}-benzoxathiol-3-yl]-3-methylphenol Chemical compound CC1=CC(O)=CC=C1C1(C=2C(=CC(O)=CC=2)C)C2=CC=CC=C2S(=O)(=O)O1 OLQIKGSZDTXODA-UHFFFAOYSA-N 0.000 description 2
- 101100001475 Aeromonas hydrophila subsp. hydrophila (strain ATCC 7966 / DSM 30187 / BCRC 13018 / CCUG 14551 / JCM 1027 / KCTC 2358 / NCIMB 9240 / NCTC 8049) alr-1 gene Proteins 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- 108010077805 Bacterial Proteins Proteins 0.000 description 2
- OUYCCCASQSFEME-MRVPVSSYSA-N D-tyrosine Chemical compound OC(=O)[C@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-MRVPVSSYSA-N 0.000 description 2
- 229930195709 D-tyrosine Natural products 0.000 description 2
- 108090000698 Formate Dehydrogenases Proteins 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 2
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 2
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 2
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 2
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 101710167005 Thiol:disulfide interchange protein DsbD Proteins 0.000 description 2
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 150000001335 aliphatic alkanes Chemical class 0.000 description 2
- 150000001336 alkenes Chemical class 0.000 description 2
- 150000001371 alpha-amino acids Chemical class 0.000 description 2
- 235000008206 alpha-amino acids Nutrition 0.000 description 2
- XPNGNIFUDRPBFJ-UHFFFAOYSA-N alpha-methylbenzylalcohol Natural products CC1=CC=CC=C1CO XPNGNIFUDRPBFJ-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000011363 dried mixture Substances 0.000 description 2
- TYSBSICHVQCQDL-NKWVEPMBSA-N ethyl (2r,3s)-2-ethyl-3-hydroxybutanoate Chemical compound CCOC(=O)[C@H](CC)[C@H](C)O TYSBSICHVQCQDL-NKWVEPMBSA-N 0.000 description 2
- JPHAXHCYCMPVSY-UHFFFAOYSA-N ethyl 2-oxo-1,3-dihydroindene-1-carboxylate Chemical compound C1=CC=C2C(C(=O)OCC)C(=O)CC2=C1 JPHAXHCYCMPVSY-UHFFFAOYSA-N 0.000 description 2
- BRUOEHVDTAORQY-UHFFFAOYSA-N ethyl 4-oxo-4-phenylbutanoate Chemical compound CCOC(=O)CCC(=O)C1=CC=CC=C1 BRUOEHVDTAORQY-UHFFFAOYSA-N 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000013537 high throughput screening Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 238000001823 molecular biology technique Methods 0.000 description 2
- 229950006238 nadide Drugs 0.000 description 2
- 150000005480 nicotinamides Chemical class 0.000 description 2
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 238000000751 protein extraction Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 1
- PPKAIMDMNWBOKN-UHFFFAOYSA-N 2-Oxo-4-phenylbutyric acid Chemical compound OC(=O)C(=O)CCC1=CC=CC=C1 PPKAIMDMNWBOKN-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 description 1
- QDGMKZBNMLHUFX-UHFFFAOYSA-N 4,4-dimethyl-2-oxopentanoic acid Chemical compound CC(C)(C)CC(=O)C(O)=O QDGMKZBNMLHUFX-UHFFFAOYSA-N 0.000 description 1
- 102000005369 Aldehyde Dehydrogenase Human genes 0.000 description 1
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000008000 CHES buffer Substances 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- XDWQYMXQMNUWID-UHFFFAOYSA-N Ethyl 2-benzylacetoacetate Chemical compound CCOC(=O)C(C(C)=O)CC1=CC=CC=C1 XDWQYMXQMNUWID-UHFFFAOYSA-N 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-N Formic acid Chemical compound OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- MKWKNSIESPFAQN-UHFFFAOYSA-N N-cyclohexyl-2-aminoethanesulfonic acid Chemical compound OS(=O)(=O)CCNC1CCCCC1 MKWKNSIESPFAQN-UHFFFAOYSA-N 0.000 description 1
- 239000007990 PIPES buffer Substances 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 239000004280 Sodium formate Substances 0.000 description 1
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 description 1
- 241000222124 [Candida] boidinii Species 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 101150063729 alr1 gene Proteins 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 229960005261 aspartic acid Drugs 0.000 description 1
- 238000011914 asymmetric synthesis Methods 0.000 description 1
- 150000001576 beta-amino acids Chemical class 0.000 description 1
- 239000006177 biological buffer Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- 125000002843 carboxylic acid group Chemical group 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000006114 decarboxylation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- QPMJENKZJUFOON-PLNGDYQASA-N ethyl (z)-3-chloro-2-cyano-4,4,4-trifluorobut-2-enoate Chemical compound CCOC(=O)C(\C#N)=C(/Cl)C(F)(F)F QPMJENKZJUFOON-PLNGDYQASA-N 0.000 description 1
- FGSGHBPKHFDJOP-UHFFFAOYSA-N ethyl 2-oxocyclohexane-1-carboxylate Chemical compound CCOC(=O)C1CCCCC1=O FGSGHBPKHFDJOP-UHFFFAOYSA-N 0.000 description 1
- PWRUKIPYVGHRFL-UHFFFAOYSA-N ethyl 3-oxo-2-phenylbutanoate Chemical compound CCOC(=O)C(C(C)=O)C1=CC=CC=C1 PWRUKIPYVGHRFL-UHFFFAOYSA-N 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 150000002211 flavins Chemical class 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 238000012188 high-throughput screening assay Methods 0.000 description 1
- 238000007871 hydride transfer reaction Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 235000011181 potassium carbonates Nutrition 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 235000017550 sodium carbonate Nutrition 0.000 description 1
- HLBBKKJFGFRGMU-UHFFFAOYSA-M sodium formate Chemical compound [Na+].[O-]C=O HLBBKKJFGFRGMU-UHFFFAOYSA-M 0.000 description 1
- 235000019254 sodium formate Nutrition 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 238000011916 stereoselective reduction Methods 0.000 description 1
- 125000000547 substituted alkyl group Chemical group 0.000 description 1
- 125000003107 substituted aryl group Chemical group 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- WUUHFRRPHJEEKV-UHFFFAOYSA-N tripotassium borate Chemical compound [K+].[K+].[K+].[O-]B([O-])[O-] WUUHFRRPHJEEKV-UHFFFAOYSA-N 0.000 description 1
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
- C12N9/0018—Phenylalanine dehydrogenase (1.4.1.20)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
- C12Y104/01009—Leucine dehydrogenase (1.4.1.9)
Definitions
- Enzymes are proteins that are capable of catalyzing chemical transformations. Enzymes position a substrate or substrates in an optimal configuration and stabilize the transition state in the reaction pathway, thereby determining which of several potential chemical transformations actually occurs. Enzymes can be highly specific, both in terms of the reaction that occurs and in their choice of substrate. Enzymes often accelerate reactions by factors of more than a million. Because of their specificity and catalytic power, enzymes are increasingly being used for industrial applications.
- Oxidoreductases catalyze redox reactions, such as the reduction of aldehydes and ketones to alcohols, the reductive amination of ketones, aldehydes, and ketoacids to amines and amino acids, the reduction of disulfides to thiols, the reduction of alkenes to alkanes and the like. These reactions are normally reversible, and frequently the same enzymes catalyze the corresponding oxidation reactions.
- alcohol dehydrogenases and carbonyl reductases catalyze both the reduction of aldehydes and ketones to alcohols and the oxidation of alcohols to aldehydes and ketones.
- Amino acid dehydrogenases catalyze the oxidation of amino acids to 2-ketoacids and the reductive amination of 2-ketoacids in the presence of ammonium salts to amino acids.
- disulfide reductases catalyze the oxidation of thiols to disulfides or mixed disulfides. Reduction and oxidation reactions are collectively referred to herein as “redox reactions.”
- Oxidoreductases can be used to produce fine and specialty chemicals, and are especially useful for producing chiral intermediates in the pharmaceutical and agricultural industries. Oxidoreductases, like many other enzymes, require other molecules, such as cofactors and cosubstrates, for optimal activity. For example, mixed function oxidases use nicotinamide cofactors as part of the complex catalysis of a hydroxylation reaction for the production of chiral alcohols.
- ketones catalyzed by alcohol dehydrogenases, ketoreductases and carbonyl reductases is known for certain ketones, but enzymes are not available for catalyzing this reaction with many desired target ketones.
- Transaminases are known that catalyze the transamination of many 2-ketoacids to alpha-amino acids, but certain target 2-ketoacids, particularly those corresponding to non-naturally occurring amino acids, are transaminated poorly, if at all.
- the most common method of detecting enzymes using nicotinamide cofactors involves the direct measurement of the cofactor.
- the concomitant oxidation of reduced nicotinamide i.e., the conversion of a reduced form of nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide (NAD + ) or the conversion of a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide phosphate (NADP + ) can be detected using the absorbance of the reduced form of the cofactor.
- NADH nicotinamide adenine dinucleotide
- NAD + the conversion of a reduced form of nicotinamide adenine dinucleotide phosphate
- NADP + nicotinamide adenine dinucleotide phosphate
- This reaction of the cofactor can be monitored in a solution by observing the decrease in the absorbance of the solution at 340 nm using a spectrophotometer.
- NAD + is converted to NADH or NADP + is converted to NADPH.
- the reduction of the cofactor can be detected by monitoring the increase in absorbance at 340 nm, corresponding to the increase in concentration of reduced nicotinamide cofactor.
- fluorescence measurements of nicotinamide cofactors can be performed as well.
- the change in concentration of oxidized or reduced nicotinamide cofactor can be used to detect other enzymes catalyzing cofactor-requiring reactions of interest.
- Detection of enzymatic activity is often performed on many enzyme sources for a particular reaction of interest in a process called screening.
- screening and particularly when carrying out high throughput screening, mixtures of cells or cell lysates containing suspended insoluble material are used as potential sources for new enzymes because clarification of the crude mixtures is operationally difficult.
- the difficulty in using such crude mixtures for routine screening for nicotinamide cofactor-using enzymes is that the reaction mixtures contain suspended solid material in the form of cells or cell debris. This insoluble material impedes the transmission of light through the solution and causes high background readings in the absorbance measurements of the cofactors.
- the crude mixtures also contain various cellular metabolites and biochemicals that absorb at 340 nm, further compromising the accuracy of the measurements. These issues are even more problematic when using high throughput screening methods due to the small volumes used in high density array formats such as microtiter plates or chips. Similarly, if fluorescence measurements are carried out, detecting the emission of fluorescence is also impeded by the presence of insoluble material.
- the products of the desired enzymatic reaction can be detected directly by chromatographic techniques.
- This method requires sampling each individual reaction followed by chromatographic separation of the reaction products, which may include alcohols, carbonyl compounds, and the like.
- Such a procedure is complex and time-consuming and is impractical for high throughput screening assays when many enzyme sources are tested for the desired enzymatic activity.
- the present invention provides novel methods for chemically transforming compounds using a mutated enzyme.
- the invention is directed to a method for the production of an amino acid from a target 2-ketoacid.
- the method comprises creating a mutated enzyme that catalyzes the reductive amination or transamination of the target 2-ketoacid; and providing the mutated enzyme in a reaction mixture comprising the target 2-ketoacid under conditions sufficient to permit the formation of the amino acid to thereby produce the amino acid.
- the invention is directed to a method for the production of an amine from a target ketone.
- the method comprises creating a mutated enzyme that catalyzes the reductive amination or transamination of the target ketone; and providing the mutated enzyme in a reaction mixture comprising the target ketone under conditions sufficient to permit the formation of the amine to thereby produce the amine.
- the invention is directed to a method for the production of an alcohol from a target ketone.
- the method comprises creating a mutated enzyme that catalyzes the reduction of the target ketone; and providing the mutated enzyme in a reaction mixture comprising the target ketone under conditions sufficient to permit the formation of the alcohol to thereby produce the alcohol.
- the mutated enzyme may be created by providing an existing enzyme and mutating the existing enzyme to produce the mutated enzyme.
- the activity of the mutated enzyme on the target 2-ketoacid or ketone is determined by contacting the mutated enzyme with a composition comprising the target 2-ketoacid or ketone and thereafter determining whether there is a change in the pH of the composition. Thereafter, it is determined whether the mutated enzyme has more activity than the existing enzyme on the target 2-ketoacid or ketone.
- the existing enzyme and/or mutated enzyme can be present in a composition containing whole cells, cell extracts, cell lysates, mixtures containing insoluble cells, particulates, cellular debris, or the like.
- compositions that also absorb light at 340 nm do not interfere with the detection of enzymatic activity using the method of the present invention because the pH change can be determined, for example, by observing a color change at a different wavelength. Further, the wavelength of the color change can be selected by using an appropriate pH indicator.
- the present invention is directed to methods for chemically transforming compounds using a mutated enzyme.
- the invention is directed to a method for the production of an amino acid from a target 2-ketoacid, the production of an amine from a target ketone and the production of an alcohol from a target ketone.
- the inventive method comprises creating a mutated enzyme that catalyzes the reductive amination or transamination of the target 2-ketoacid or ketone or the reduction of the target ketone and providing the mutated enzyme in a reaction mixture comprising the target 2-ketoacid or ketone under conditions sufficient to permit the formation of the desired amino acid, amine or alcohol to thereby produce the amino acid, amine or alcohol.
- mutated and “mutating” refer broadly to any of a variety of molecular biology techniques, such as mutagenesis, shuffling, molecular breeding, and gene reassembly, that can be used to create vast numbers of mutant versions of an enzyme encoded by a known gene.
- the activity of the mutated enzyme on the target compound is determined by contacting the mutated enzyme with a composition comprising the target compound and thereafter determining whether there is a change in the pH of the composition. Thereafter it is determined whether the mutated enzyme has more activity than the existing enzyme on the target compound.
- Non-limiting examples of sources of material that can be screened to obtain the existing enzyme include microorganisms, such as bacteria and yeast, which naturally express oxidoreductases, and genetically modified microorganisms, which express wild-type, modified or mutated oxidoreductases.
- useful materials to be screened include cell lysates, mixtures of cells, cell extracts, environmental samples and isolates, and the like.
- the material may be provided as a solution, a suspension, a dried mixture, a solid, or the like.
- the composition to be screened may be prepared and stored as a solution or as a suspension in liquid form. The composition may be maintained at room temperature, at refrigerator temperatures, or frozen.
- the composition may be prepared by lyophilization or evaporation of a liquid composition.
- the solid composition may be prepared by mixing solid ingredients such as a cofactor, a pH indicator, and a target compound.
- solid ingredients such as a cofactor, a pH indicator, and a target compound.
- target compound refers to a substance that is desired to be acted upon by an enzyme as a substrate.
- Typical target compounds include aldehydes, ketones, disulfides, thiols, ketoacids, amines, amino acids, alcohols, alkenes, alkanes, and the like.
- target compound does not include enzyme substrates that undergo a hydrolytic transformation that results in the creation or removal of an acidic or basic functionality, such as a carboxylic acid group.
- Target compounds are often chiral and/or transformed into chiral compounds by enzymes, and enrichment in single stereoisomers can occur.
- pH indicator means any material or substance that changes its properties in response to a change in pH. Preferred changes in properties include a change in optical properties, such as a color change.
- pH indicators useful in the practice of the present invention include, but are not limited to, cresol red, m-cresol purple, bromothymol blue, bromophenol red, bromophenol blue, phenol red, and phenolphthalein.
- the pH indicator can be selected independently for each screen to determine the pH range or match a desired pH range for the enzyme to be detected. For example, m-cresol purple is yellow at a pH of about 7.4 and purple at a pH of about 9.0.
- Cresol red is yellow at a pH of about 7.2 and red at a pH of about 8.8.
- Bromothymol blue is yellow at a pH of about 6.0 and red at a pH of about 7.6.
- Bromophenol red is yellow at a pH of about 5.2 and red at a pH of about 6.8.
- Bromophenol blue is yellow at a pH of about 3.0 and blue at a pH of about 4.6.
- Phenol red is yellow at a pH of about 6.8 and red at a pH of about 8.2.
- Phenolphthalein is colorless at a pH of about 8.0 and pink at a pH of about 9.8.
- Other pH indicators can be selected depending on the desired pH range for the reaction and the desired color change.
- the conditions of the determination step can be adjusted to favor the detection or screening of an enzyme with a desired pH optimum by adjusting the pH of the reaction mixture used in the screen.
- the reagent composition used for the screen can be buffered at a pH of 6 using a buffer that has its optimum buffering capacity near pH 6, and a pH indicator can be selected that changes color within the range of pH 5 to 7.
- the reagent composition used for the screen can be buffered at a pH of 9 using a buffer that has its optimum buffering capacity near pH 9, and a pH indicator can be selected that changes color within the range of pH 8 to 10.
- the pH indicator is selected such that it exhibits a color change in response to a change in pH within a range of about 1 to 1.5 pH units on either side of the desired pH for the reaction.
- the concentration of the buffer is preferably adjusted in order to maintain a desired initial pH for the screening reaction mixture and to reduce or eliminate small changes in pH not caused by the desired redox reaction.
- concentration of the buffer should not be so high as to impede the change of pH that occurs as the reaction catalyzed by the oxidoreductase proceeds.
- the buffer may be any substance that helps maintain the desired initial pH of the solution. Examples include potassium phosphate, sodium phosphate, potassium borate, sodium borate, sodium acetate, potassium acetate, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, TRIS, PIPES, HEPES, MOPS, TEA, CHES, and the like.
- buffer concentrations using the method of the present invention are from about 0.01 mM to about 20 mM, and preferably from about 0.05 mM to about 5 mM.
- oxidoreductase refers to an enzyme capable of performing an oxidation reaction or reduction reaction.
- Nonlimiting examples of oxidoreductases include a reductase, an oxidase, a dehydrogenase, a ketoreductase, an alcohol dehydrogenase, a carbonyl reductase, an aldehyde dehydrogenase, an amino acid dehydrogenase, an amine oxidase, a disulfide reductase, an enoate reductase, and a mixed function oxidase.
- a listing of such enzymes can be found in Enzyme Nomenclature ; Webb, E. C., Ed. Academic: Orlando 1984; pp 20-141, the disclosure of which is incorporated herein by reference.
- cofactor means any molecule that participates in a chemical transformation of the target compound, including cofactors and cosubstrates.
- cofactors include nicotinamide cofactors, flavins, and derivatives and analogs thereof.
- nicotinamide cofactor refers to any type of the oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD + and NADH, respectively) and the oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate (NADP + and NADPH, respectively) and derivatives and analogs thereof.
- derivative means any compound containing a pyridine structural element, including nicotinamides that have been chemically modified by attachment to soluble or insoluble polymeric materials.
- analogs refers to materials that undergo a formal hydride transfer in a redox reaction similar to that undergone by nicotinamide cofactors.
- analogs of nicotinamide cofactors useful in the practice of the present invention include compounds described in U.S. Pat. No. 5,801,006, the disclosure of which is incorporated herein by reference.
- Other suitable cofactors, as defined herein, can be used in the practice of the invention, as would be recognized by those skilled in the art.
- the nicotinamide cofactors can be used in equimolar quantities relative to the target ketone, alcohol, amine or amino acid, or the cofactors may be recycled, if desired.
- Numerous methods for the recycling of nicotinamide cofactors are well-known in the art, and any of these methods may be used in the practice of the present invention.
- Some of the methods for recycling nicotinamide cofactors are described in G. L. Lemiere, et al., Tetrahedron Letters, 26, 4257 (1985); in “Enzymes as Catalysts for Organic Synthesis,” pp. 19-34, M. Schneider, Ed., Reidel Dordecht, 1986; in Z.
- an amount of about 0.0001 mole to about 0.05 mole of nicotinamide cofactor is used per mole of ketone to be reduced or reductively aminated, per mole of 2-ketoacid to be reductively aminated, or per mole of alcohol or amine or amino acid to be oxidized, providing a recycle number for the cofactor of from about 20 to about 10,000.
- a and B are independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocyclic, and the like.
- one proton is consumed in the reaction for each molecule of nicotinamide cofactor oxidized and each molecule of alcohol formed.
- the consumption of protons causes the pH of the reaction mixture to rise.
- a suitable pH indicator in the reaction mixture, the presence of an alcohol dehydrogenase is indicated by a change in color of the reaction mixture.
- positive reactions can be detected spectrophotometrically, if desired, the use of a colorimetric pH indicator has the added advantage that the presence of oxidoreductase enzymes can be detected visually and without expensive instrumentation.
- an oxidation reaction can also be used for screening and detection.
- an alcohol dehydrogenase or carbonyl reductase catalyzes the oxidation of an alcohol to form an oxidized carbonyl compound, shown in Scheme 2.
- the presence of an oxidoreductase catalyzing this reaction can be detected using a reaction mixture containing an oxidized nicotinamide cofactor, an alcohol, and a pH indicator.
- the pH of the reaction mixture will decrease as the reaction progresses, and the decrease in pH is detected by the change in color of the pH indicator.
- carbonyl compound means any chemical compound that has incorporated into it a functional group consisting of a carbon-oxygen double bond.
- carbonyl reductase means any enzyme that can catalyze the chemical reduction of a carbonyl group in the presence of a nicotinamide cofactor.
- ammonia or ammonium ion is also a reactant.
- ammonia or a salt of ammonium ion is also included with a pH indicator, a reduced nicotinamide cofactor, and a ketone or ketoacid to be reductively aminated.
- the reaction is shown in Scheme 3.
- the reaction mixture for detection contains a pH indicator, an oxidized nicotinamide cofactor, and an amine or amino acid to be oxidized.
- the reaction for the oxidation of an amine or amino acid using an amine or amino acid dehydrogenase is depicted in Scheme 4:
- the above-described method can be used to detect enzymes using a second reaction that can be coupled to the enzyme-catalyzed reaction to be screened.
- the reaction products are an amine or amino acid and oxaloacetate.
- the oxaloacetate can be decarboxylated to pyruvate, with the consumption of a proton.
- aminotransferase activity can be detected by detecting an increase in the pH of the reaction mixture because the decarboxylation of oxaloacetate is coupled to the transamination reaction to be screened.
- This method is particularly useful for screening for enzymes to perform specific chemical transformations of target compounds that are intermediates in chemical syntheses.
- an enzyme after an enzyme has been determined to have activity for a particular target compound, it can be used to convert that target compound to a useful chemical intermediate, as described above.
- useful chemical intermediates include alcohols, amines, alpha-amino acids, beta-amino acids, gamma-amino acids, aldehydes, ketones, carboxylic acids, esters, amides, and the like.
- a cofactor is present with the enzyme. Suitable cofactors are set forth above.
- the target compound is a ketone that is not a ketoacid
- the target compound is converted to an amine, preferably a chiral amine, in the presence of an amine dehydrogenase.
- the above-described screening method is used to first determine enzymatically-active amino acid dehydrogenases. Further screening is then performed on the enzymatically-active amino acid dehydrogenases, again, in accordance with the procedures described above, to identify enzymatically-active amine dehydrogenases. The thus identified enzymatically-active amine dehydrogenase is then provided in a solution together with a ketone, ammonia and reduced nicotinamide cofactor to synthesize an amine.
- a gene encoding the alcohol dehydrogenase YPR1 (described by Nakamura, K., et al., Bioscience, Biotechnology and Biochemistry , (1997) 61, 375-377), is subjected to mutagenesis by error-prone PCR according to the method of May, O., et al., ( Nature Biotechnology , (2000) 18, 317-320).
- the error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as a template dissolved in PCR buffer (10 mM TRIS, 1.5 mM MgCl 2 , 50 mM KCl, pH 8.3), and also containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase (Roche Diagnostics, Indianapolis, Ind.).
- Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C.
- the PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector (Invitrogen, Carlsbad, Calif.) which has been digested with the same restriction enzymes.
- the resulting YPR 1 mutant library is transformed into the E. coli host strain LMG 194 (Invitrogen, Carlsbad, Calif.) and plated on LB agar supplied with 100 ⁇ g/mL ampicillin. Individual transformants are inoculated into 96-well microtiter plates (hereafter referred to as master plates) containing 0.2 mL LB Broth with 100 ⁇ g/mL ampicillin, and growth is allowed to take place for 8 to 16 hours at 37° C.
- each well in each master plate is then re-inoculated by a replica plating technique into a new second stage 96-well plate pre-loaded with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5-10 hours at 37° C. with shaking at 200 rpm.
- the second stage plates are then centrifuged at 14,000 rpm for 20 minutes, and the supernatant is decanted. The cell pellet in each well is washed with 200 mL of water.
- B-Per Bacterial Protein Extraction Reagent (Pierce Chemical Co., Rockford, Ill.), hereinafter “B-Per.” After mixing, the suspension of cells in B-Per reagent is allowed to stand for 10 minutes at room temperature, and a reaction solution having the following composition is then added to each well in the plate:
- Wells containing an alcohol dehydrogenase that catalyzes the reduction of the target compound ethyl-4-chloro-3-ketobutyrate can be identified easily as their color changes from an initial yellow to an orange or red color. These wells are correlated to the original wells in the master plates to obtain the original clones of mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- Example 1 The procedure of Example 1 is repeated, replacing the ethyl-4-chloro-3-ketobutyrate with ethyl-3-phenyl-3-ketopropionate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- Example 1 The procedure of Example 1 is repeated, replacing the ethyl-4-chloro-3-ketobutyrate with ethyl-indan-2-one-1-carboxylate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- Example 1 The procedure of Example 1 is repeated, replacing the ethyl-4-chloro-3-ketobutyrate with ethyl-4-phenyl-4-ketobutyrate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- Example 1 The procedure of Example 1 is repeated, replacing the reaction solution with a reaction solution of the following composition:
- Wells containing an alcohol dehydrogenase that reduces ethyl-3-phenyl-3-ketopropionate can be identified easily as the color changes from an initial yellow to a red color. At least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction is identified.
- Example 5 The procedure of Example 5 is repeated, replacing the ethyl-3-phenyl-3-ketopropionate with ethyl-indan-2-one-1-carboxylate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- Example 5 The procedure of Example 5 is repeated, replacing the ethyl-3-phenyl-3-ketopropionate with ethyl-4-phenyl-4-ketobutyrate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- Example 5 The procedure of Example 5 is repeated, replacing the ethyl-3-phenyl-3-ketoproponiate with ethyl-cyclohexanone-2-carboxylate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- Example 1 The procedure of Example 1 is repeated, replacing the reaction solution with a reaction solution of the following composition:
- Example 9 The procedure of Example 9 is repeated, replacing the ethyl-2-ethyl-3-ketobutyrate with ethyl-2-allyl-3-ketobutyrate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- Example 9 The procedure of Example 9 is repeated, replacing the ethyl-2-ethyl-3-ketobutyrate with ethyl-2-phenyl-3-ketobutyrate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- Example 9 The procedure of Example 9 is repeated, replacing the ethyl-2-ethyl-3-ketobutyrate with ethyl-2-benzyl-3-ketobutyrate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- a gene encoding the alcohol dehydrogenase Alr1 (Yamada, et al., FEMS Microbiology Letters , (1990) 70, 45-48) is subjected to mutagenesis by error-prone PCR according to the method of May et al.
- the error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PCR buffer containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase.
- Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C.
- the PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector which has been digested with the same restriction enzymes.
- the resulting Alr1 mutant library is transformed into an E. coli host strain LMG194 and plated on LB agar supplied with 100 ⁇ g/mL ampicillin. Individual transformants are inoculated into master plates containing 0.2 mL LB Broth with 100 ⁇ g/mL ampicillin, and growth is allowed to take place for 8-16 hours at 37° C. with shaking at 200 rpm.
- Each well in each master plate is then re-inoculated by a replica plating technique into a new second stage 96-well plate pre-loaded with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5 to 10 hours at 37° C. with shaking at 200 rpm.
- the second stage plates are then centrifuged at 14,000 rpm for 20 minutes, and the supernatant is decanted.
- the cell pellet in each well is washed with 200 mL of water.
- the washed cell pellet is suspended in 30 mL of B-Per. After mixing, the suspension of cells in B-Per reagent is allowed to stand for 10 minutes at room temperature, and a solution having the following composition is then added to each well in the plate:
- Wells in which the color changes from an initial blue to a yellow color contain mutant alcohol dehydrogenases that catalyze the oxidation of the target alcohol (2R,3S)-ethyl-2-ethyl-3-hydroxybutyrate. These wells are correlated to the original wells in the master plates to obtain the original clones of mutant alcohol dehydrogenases catalyzing the desired oxidation reaction.
- a gene encoding leucine dehydrogenase from B. stearothermophilus (Nagata, et al. Biochemistry (1998) 27, 9056) is subjected to mutagenesis by error-prone PCR according to the method of May et al.
- the error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PCR buffer also containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase. Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C.
- the PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector which has been digested with the same restriction enzymes.
- the resulting leucine dehydrogenase mutant library is transformed into an E. coli host strain LMG194 and plated on LB agar supplied with 100 ⁇ g/mL ampicillin. Individual transformants are inoculated into master plates containing 0.2 mL LB Broth with 100 ⁇ g/mL ampicillin, and growth is allowed to take place for 8 to 16 hours at 37° C. with shaking at 200 rpm.
- Each well in each master plate is then re-inoculated by a replica plating technique into a new second stage 96-well plate pre-loaded with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5-10 hours at 37° C. with shaking at 200 rpm.
- the second stage plates are then centrifuged at 14,000 rpm for 20 minutes, and the supernatant is decanted.
- the cell pellet in each well is washed with 200 mL of water.
- the washed cell pellet is suspended in 30 mL of B-Per After mixing, the suspension of cells in B-Per reagent is allowed to stand for 10 minutes at room temperature, and a solution having the following composition is then added to each well in the plate:
- Wells in which the color changes from an initial yellow to an orange or red color contain leucine dehydrogenase that catalyzes the reductive amination of the target 2-ketoacid 3,3-dimethyl-2-ketobutyrate. These wells are correlated to the original wells in the master plates to obtain the original clones of mutant leucine dehydrogenase that catalyzes the desired reductive amination reaction.
- Example 14 The procedure of Example 14 is repeated, replacing 3,3 -dimethyl-2-ketobutyrate with 4-(methylphosphinyl)-2-ketobutyrate, thereby identifying at least one mutant dehydrogenase that catalyzes the desired reductive amination reaction.
- Example 14 The procedure of Example 14 is repeated, replacing 3,3-dimethyl-2-ketobutyrate with 3-(2-naphthyl)pyruvate, thereby identifying at least one mutant dehydrogenase that catalyzes the desired reductive amination reaction.
- Example 14 The procedure of Example 14 is repeated, replacing the 3,3-dimethyl-2-ketobutyrate with 3-(1-naphthyl)pyruvate, thereby identifying at least one mutant dehydrogenase that catalyzes the desired reductive amination reaction.
- Example 14 The procedure of Example 14 is repeated, replacing 3,3-dimethyl-2-ketobutyrate with 4-phenyl-2-ketobutyrate, thereby identifying at least one mutant dehydrogenase that catalyzes the desired reductive amination reaction.
- Example 14 The procedure of Example 14 is repeated replacing the 3,3-dimethyl-2-ketobutyrate with 4,4-dimethyl-2-ketopentanoate, thereby identifying at least one mutant dehydrogenase that catalyzes the desired reduction reaction.
- a gene encoding the leucine dehydrogenase from B. stearothermophilus is subjected to mutagenesis by error-prone PCR according to the method of May et al.
- the error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PCR buffer (10 mM TRIS, 1.5 mM MgCl 2 , 50 mM KCl, pH 8.3), and also containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase.
- Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C.
- the PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector which has been digested with the same restriction enzymes.
- the resulting leucine dehydrogenase mutant library is transformed into an E. coli host strain LMG194 and plated on LB agar supplied with 100 ⁇ g/mL ampicillin.
- Wells in which the color changes from an initial blue to a yellow color contain mutant leucine dehydrogenases that catalyze the oxidation of the target amino acid. These wells are correlated to the original wells in the master plates to obtain the original clones of mutant leucine dehydrogenases catalyzing the desired oxidation reaction.
- Example 20 The procedure of Example 20 is repeated replacing the L-tert-leucine with S-phosphinothricin, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired oxidation reaction.
- Example 20 The procedure of Example 20 is repeated, replacing the L-tert-leucine with S-(2-naphthyl)alanine, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired oxidation reaction.
- Example 20 The procedure of Example 20 is repeated, replacing the L-tert-leucine with D-tert-leucine, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired oxidation reaction.
- Example 20 The procedure of Example 20 is repeated, replacing L-tert-leucine with S-4-phenyl-2-aminobutyrate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired oxidation reaction.
- Example 20 The procedure of Example 20 is repeated, replacing the L-tert-leucine with D-tyrosine, thereby identifying at lest one mutant alcohol dehydrogenase that catalyzes the desired oxidation reaction.
- a gene encoding leucine dehydrogenase from B. stearothermophilus is subjected to mutagenesis by error-prone PCR according to the method of May, et al.
- the error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PCR buffer containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase.
- Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C.
- the PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector which has been digested with the same restriction enzymes.
- the resulting leucine dehydrogenase mutant library is transformed into an E. coli host strain LMG194 and plated on LB agar supplied with 100 ⁇ g/mL ampicillin. Individual transformants are inoculated into master plates containing 0.2 mL LB Broth with 100 ⁇ g/mL ampicillin, and growth is allowed to take place for 8-16 hours at 37° C. with shaking at 200 rpm.
- Each well in each master plate is then re-inoculated by a replica plating technique into a new second stage 96-well plate pre-loaded with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5-10 hours at 37° C. with shaking at 200 rpm.
- the second stage plates are then centrifuged at 14,000 rpm for 20 minutes, and the supernatant is decanted.
- the cell pellet in each well is washed with 200 mL of water.
- the washed cell pellet is suspended in 30 mL of B-Per. After mixing, the suspension of cells in B-Per reagent is allowed to stand for 10 minutes at room temperature, and a solution having the following composition is then added to each well in the plate:
- a gene encoding the leucine dehydrogenase from B. stearothermophilus is subjected to mutagenesis by error-prone PCR according to the method of May, et al.
- the error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PCR buffer containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase.
- Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C.
- the PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector which has been digested with the same restriction enzymes.
- the resulting leucine dehydrogenase mutant library is transformed into an E. coli host strain LMG1 94 and plated on LB agar supplied with 100 ⁇ g/mL ampicillin. Individual transformants are inoculated into master plates containing 0.2 mL LB Broth with 100 ⁇ g/mL ampicillin, and growth is allowed to take place for 8-16 hours at 37° C. with shaking at 200 rpm.
- Each well in each master plate is then re-inoculated by a replica plating technique into a new second stage plate pre-loaded with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5 to 10 hours at 37° C. with shaking at 200 rpm.
- the second stage plates are then centrifuged at 14,000 rpm for 20 minutes, and the supernatant is decanted.
- the cell pellet in each well is washed with 200 mL of water.
- the washed cell pellet is suspended in 30 mL of B-Per Bacterial Protein Extraction Reagent. After mixing, the suspension of cells in B-Per reagent is allowed to stand for 10 minutes at room temperature, and a solution having the following composition is then added to each well in the plate:
- Wells in which the color changes from blue initially to yellow contain mutant leucine dehydrogenase that catalyze the oxidation of the target amine. These wells are correlated to the original wells in the master plates to obtain the original clones of mutant leucine dehydrogenases catalyzing the desired oxidation reaction.
- a gene encoding the leucine dehydrogenase from B. stearothermophilus is subjected to mutagenesis by error-prone PCR according to the method of May, et al.
- the error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PCR buffer containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase.
- Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C.
- the PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector which has been digested with the same restriction enzymes.
- the resulting leucine dehydrogenase mutant library is transformed into an E. coli host strain LMG194 and plated on LB agar supplied with 100 ⁇ g/mL ampicillin. Individual transformants are inoculated into master plates containing 0.2 mL LB Broth with 100 ⁇ g/mL ampicillin, and growth is allowed to take place for 8-16 hours at 37° C. with shaking at 200 rpm.
- Each well in each master plate is then re-inoculated by a replica plating technique into a new second stage plate pre-loaded with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5-10 hours at 37° C. with shaking at 200 rpm.
- the second stage plates are then centrifuged at 14,000 rpm for 20 minutes, and the supernatant is decanted.
- the cell pellet in each well is washed with 200 mL of water.
- the washed cell pellet is suspended in 30 mL of B-Per. After mixing, the suspension of cells in B-Per reagent is allowed to stand for 10 minutes at room temperature, and a solution having the following composition is then added to each well in the plate:
- Wells in which the color changes from blue initially to yellow contain mutant leucine dehydrogenase that catalyze the oxidation of the target amine. These wells are correlated to the original wells in the master plates to obtain the original clones of mutant leucine dehydrogenases catalyzing the desired oxidation reaction.
- One hundred units of an amine dehydrogenase generated by mutagenesis and screening of leucine dehydrogenase as described in any one of Examples 26 to 28 above is incubated at 45° C. in 100 milliliters of a solution maintained at pH 6.5 containing potassium phosphate (1 millimole), NADH (0.01 millimole), ammonium formate (25 millimoles), and formate dehydrogenase from Candida boidinii (100 units). Acetophenone (10 millimoles) is added slowly over one hour with stirring, and the reaction is allowed to proceed for an additional 4 hours.
- reaction products After basification of the reaction mixture to pH 12 and extraction with methyl t-butyl ether, analysis of the reaction products is carried out by gas chromatography to determine the yield of 1-phenylethylamine. Chiral analysis is carried out by chiral gas chrmoatography using a ChiraDex CB column (Advanced Separation Technology, Whippany, N.J. USA).
- Example 29 The method of Example 29 is carried out except that the amine dehydrogenase is an R-1-phenylethylamine dehydrogenase and the product is R-1-phenylethylamine.
- Example 29 The method of Example 29 is carried out except that the amine dehydrogenase is an S-1-phenylethylamine dehydrogenase and the product is S-1-phenylethylamine.
- Example 30 The method of Example 30 is carried out except that acetophenone is replaced by p-chloroacetophenone and the product is R-1-(p-chlorophenyl)ethylamine.
- Example 31 The method of Example 31 is carried out except that acetophenone is replaced by p-chloroacetophenone and the product is S-1-(p-chlorophenyl)ethylamine.
- Example 30 The method of Example 30 is carried out except that acetophenone is replaced by m-bromoacetophenone and the product is R-1-(m-bromophenyl)ethylamine.
- Example 31 The method of Example 31 is carried out except that acetophenone is replaced by m-bromoacetophenone and the product is S-1-(m-bromophenyl)ethylamine.
- the reaction mixture is extracted with methyl t-butyl ether, and analysis of the reaction products is carried out by gas chromatography to determine the yield of 1-phenylethanol. Chiral analysis is carried out by chiral gas chromatography using a ChiraDex CB column (Advanced Separation Technology, Whippany, N.J. USA).
- Example 36 The method of Example 36 is carried out except that the alcohol dehydrogenase is determined to be an R-1-phenylethanol dehydrogenase and the product is R-1-phenylethanol.
- Example 36 The method of Example 36 is carried out except that the alcohol dehydrogenase is determined to be an 5-1-phenylethanol dehydrogenase and the product is S-1-phenylethanol,
- Example 36 The method of Example 36 is carried out except that acetophenone is replaced by p-chloroacetophenone, the alcohol dehydrogenase is determined to be an R-1-(p-chlorophenyl)ethanol dehydrogenase and the product is R-1-(p-chlorophenyl)ethanol.
- Example 36 The method of Example 36 is carried out except that acetophenone is replaced by p-chloroacetophenone, the alcohol dehydrogenase is determined to be an S-1-(p-chlorophenyl)ethanol dehydrogenase and the product is S-1-(p-chlorophenyl)ethanol.
- Example 36 The method of Example 36 is carried out except that acetophenone is replaced by m-bromoacetophenone, the alcohol dehydrogenase is determined to be an R-1-(m-bromophenyl)ethanol dehydrogenase, and the product is R-1-(m-bromophenyl)ethanol.
- Example 36 The method of Example 36 is carried out except that acetophenone is replaced by m-bromoacetophenone, the alcohol dehydrogenase is determined to be an S-1-(m-bromophenyl)ethanol dehydrogenase, and the product is S-1-(m-bromophenyl)ethanol.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
- This application is a continuation-in-part of U.S. application Ser. No. 09/702,421, filed Oct. 31, 2000, and claims the benefit of U.S. Provisional Application No. 60/288,378, filed May 3, 2001, the entire disclosures of which are incorporated herein by reference.
- Enzymes are proteins that are capable of catalyzing chemical transformations. Enzymes position a substrate or substrates in an optimal configuration and stabilize the transition state in the reaction pathway, thereby determining which of several potential chemical transformations actually occurs. Enzymes can be highly specific, both in terms of the reaction that occurs and in their choice of substrate. Enzymes often accelerate reactions by factors of more than a million. Because of their specificity and catalytic power, enzymes are increasingly being used for industrial applications.
- One family of enzymes that is especially useful for industrial applications is the family of oxidoreductase enzymes. Oxidoreductases catalyze redox reactions, such as the reduction of aldehydes and ketones to alcohols, the reductive amination of ketones, aldehydes, and ketoacids to amines and amino acids, the reduction of disulfides to thiols, the reduction of alkenes to alkanes and the like. These reactions are normally reversible, and frequently the same enzymes catalyze the corresponding oxidation reactions. For example, alcohol dehydrogenases and carbonyl reductases catalyze both the reduction of aldehydes and ketones to alcohols and the oxidation of alcohols to aldehydes and ketones. Amino acid dehydrogenases catalyze the oxidation of amino acids to 2-ketoacids and the reductive amination of 2-ketoacids in the presence of ammonium salts to amino acids. Similarly, disulfide reductases catalyze the oxidation of thiols to disulfides or mixed disulfides. Reduction and oxidation reactions are collectively referred to herein as “redox reactions.”
- Some oxidoreductases can be used to produce fine and specialty chemicals, and are especially useful for producing chiral intermediates in the pharmaceutical and agricultural industries. Oxidoreductases, like many other enzymes, require other molecules, such as cofactors and cosubstrates, for optimal activity. For example, mixed function oxidases use nicotinamide cofactors as part of the complex catalysis of a hydroxylation reaction for the production of chiral alcohols.
- Although a number of different enzymes are known, the development of new applications for enzymes such as oxidoreductases requires an expanded search for new enzymes that catalyze specific reactions of interest. For example, amino acid dehydrogenases that reductively aminate certain 2-ketoacids to naturally occurring L-amino acids are known, but no suitable amino acid dehydrogenase has been identified for the production of many non-naturally occurring amino acids. The enzyme catalyzed reductive amination of ketones that are not 2-ketoacids is comparatively quite rare. Similarly, the stereoselective reduction of ketones catalyzed by alcohol dehydrogenases, ketoreductases and carbonyl reductases is known for certain ketones, but enzymes are not available for catalyzing this reaction with many desired target ketones. Transaminases are known that catalyze the transamination of many 2-ketoacids to alpha-amino acids, but certain target 2-ketoacids, particularly those corresponding to non-naturally occurring amino acids, are transaminated poorly, if at all.
- There are several known methods to generate potential enzymes that catalyze specific reactions of interest. For example, diverse populations of enzymes can be found in microorganisms harvested from different environments. These microorganisms can be cultured, and their DNA extracted, amplified by PCR, and cloned into a host for expression of the enzymes. Alternatively, various molecular biology techniques, such as mutagenesis, shuffling, molecular breeding, and gene reassembly, can be used to create vast numbers of mutant versions of an enzyme encoded by a known gene. Examples of gene shuffling and molecular breeding are described in U.S. Pat. No.5,605,793; U.S. Pat. No. 5,811,238; U.S. Pat. No. 5,830,721; U.S. Pat. No. 5,837,458; U.S. Pat. No. 5,965,408; U.S. Pat. No. 5,958,672; U.S. Pat. No. 6,001,574; and U.S. Pat. No. 6,117,679, all incorporated herein by reference. Examples of methods for constructing large numbers of mutants are described in U.S. Pat. No.6,001,574; U.S. Pat. No.6,030,779; and U.S. Pat. No.6,054,267, also incorporated herein by reference.
- Once potential enzymes that may be able to catalyze specific reactions of interest have been generated, the enzymes are tested for activity on the desired substrate, or target compound. Because many enzymes such as oxidoreductases require nicotinamide cofactors for optimal activity, detection of the oxidation or reduction of the cofactor can be used as a signal of enzyme activity.
- Currently, the most common method of detecting enzymes using nicotinamide cofactors involves the direct measurement of the cofactor. For example, as a carbonyl reductase reduces a carbonyl group, the concomitant oxidation of reduced nicotinamide, i.e., the conversion of a reduced form of nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide (NAD+) or the conversion of a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide phosphate (NADP+) can be detected using the absorbance of the reduced form of the cofactor. This reaction of the cofactor can be monitored in a solution by observing the decrease in the absorbance of the solution at 340 nm using a spectrophotometer. Alternatively, during the carbonyl reductase-catalyzed oxidation of an alcohol to the corresponding aldehyde or ketone, NAD+ is converted to NADH or NADP+ is converted to NADPH. The reduction of the cofactor can be detected by monitoring the increase in absorbance at 340 nm, corresponding to the increase in concentration of reduced nicotinamide cofactor. Similarly, fluorescence measurements of nicotinamide cofactors can be performed as well. Additionally, the change in concentration of oxidized or reduced nicotinamide cofactor can be used to detect other enzymes catalyzing cofactor-requiring reactions of interest.
- Detection of enzymatic activity is often performed on many enzyme sources for a particular reaction of interest in a process called screening. Often when screening, and particularly when carrying out high throughput screening, mixtures of cells or cell lysates containing suspended insoluble material are used as potential sources for new enzymes because clarification of the crude mixtures is operationally difficult. The difficulty in using such crude mixtures for routine screening for nicotinamide cofactor-using enzymes is that the reaction mixtures contain suspended solid material in the form of cells or cell debris. This insoluble material impedes the transmission of light through the solution and causes high background readings in the absorbance measurements of the cofactors. The crude mixtures also contain various cellular metabolites and biochemicals that absorb at 340 nm, further compromising the accuracy of the measurements. These issues are even more problematic when using high throughput screening methods due to the small volumes used in high density array formats such as microtiter plates or chips. Similarly, if fluorescence measurements are carried out, detecting the emission of fluorescence is also impeded by the presence of insoluble material.
- As an alternative, the products of the desired enzymatic reaction can be detected directly by chromatographic techniques. This method requires sampling each individual reaction followed by chromatographic separation of the reaction products, which may include alcohols, carbonyl compounds, and the like. Such a procedure is complex and time-consuming and is impractical for high throughput screening assays when many enzyme sources are tested for the desired enzymatic activity.
- The present invention provides novel methods for chemically transforming compounds using a mutated enzyme. In one embodiment, the invention is directed to a method for the production of an amino acid from a target 2-ketoacid. The method comprises creating a mutated enzyme that catalyzes the reductive amination or transamination of the target 2-ketoacid; and providing the mutated enzyme in a reaction mixture comprising the target 2-ketoacid under conditions sufficient to permit the formation of the amino acid to thereby produce the amino acid.
- In another embodiment, the invention is directed to a method for the production of an amine from a target ketone. The method comprises creating a mutated enzyme that catalyzes the reductive amination or transamination of the target ketone; and providing the mutated enzyme in a reaction mixture comprising the target ketone under conditions sufficient to permit the formation of the amine to thereby produce the amine.
- In yet another embodiment, the invention is directed to a method for the production of an alcohol from a target ketone. The method comprises creating a mutated enzyme that catalyzes the reduction of the target ketone; and providing the mutated enzyme in a reaction mixture comprising the target ketone under conditions sufficient to permit the formation of the alcohol to thereby produce the alcohol.
- In the above embodiments, the mutated enzyme may be created by providing an existing enzyme and mutating the existing enzyme to produce the mutated enzyme. The activity of the mutated enzyme on the target 2-ketoacid or ketone is determined by contacting the mutated enzyme with a composition comprising the target 2-ketoacid or ketone and thereafter determining whether there is a change in the pH of the composition. Thereafter, it is determined whether the mutated enzyme has more activity than the existing enzyme on the target 2-ketoacid or ketone. The existing enzyme and/or mutated enzyme can be present in a composition containing whole cells, cell extracts, cell lysates, mixtures containing insoluble cells, particulates, cellular debris, or the like. Other compounds in the composition that also absorb light at 340 nm do not interfere with the detection of enzymatic activity using the method of the present invention because the pH change can be determined, for example, by observing a color change at a different wavelength. Further, the wavelength of the color change can be selected by using an appropriate pH indicator.
- The present invention is directed to methods for chemically transforming compounds using a mutated enzyme. In particularly preferred embodiments, the invention is directed to a method for the production of an amino acid from a target 2-ketoacid, the production of an amine from a target ketone and the production of an alcohol from a target ketone. The inventive method comprises creating a mutated enzyme that catalyzes the reductive amination or transamination of the target 2-ketoacid or ketone or the reduction of the target ketone and providing the mutated enzyme in a reaction mixture comprising the target 2-ketoacid or ketone under conditions sufficient to permit the formation of the desired amino acid, amine or alcohol to thereby produce the amino acid, amine or alcohol.
- As used herein, the terms “mutated” and “mutating” refer broadly to any of a variety of molecular biology techniques, such as mutagenesis, shuffling, molecular breeding, and gene reassembly, that can be used to create vast numbers of mutant versions of an enzyme encoded by a known gene. The activity of the mutated enzyme on the target compound is determined by contacting the mutated enzyme with a composition comprising the target compound and thereafter determining whether there is a change in the pH of the composition. Thereafter it is determined whether the mutated enzyme has more activity than the existing enzyme on the target compound.
- By determining in which reactions the pH indicator undergoes a color change, enzymes with the desired enzymatic activity can be detected easily, even in a high throughput format, enabling the more facile discovery of new enzymes, particularly oxidoreductases that catalyze useful redox reactions.
- Non-limiting examples of sources of material that can be screened to obtain the existing enzyme include microorganisms, such as bacteria and yeast, which naturally express oxidoreductases, and genetically modified microorganisms, which express wild-type, modified or mutated oxidoreductases. Examples of useful materials to be screened include cell lysates, mixtures of cells, cell extracts, environmental samples and isolates, and the like. The material may be provided as a solution, a suspension, a dried mixture, a solid, or the like. As a solution or suspension, the composition to be screened may be prepared and stored as a solution or as a suspension in liquid form. The composition may be maintained at room temperature, at refrigerator temperatures, or frozen. As a solid or dried mixture, the composition may be prepared by lyophilization or evaporation of a liquid composition. Alternatively, the solid composition may be prepared by mixing solid ingredients such as a cofactor, a pH indicator, and a target compound. When a solid composition is used in the practice of the present invention, the solid composition is normally redissolved or resuspended prior to use by the addition of water or water containing buffer.
- As used herein, “target compound” refers to a substance that is desired to be acted upon by an enzyme as a substrate. Typical target compounds include aldehydes, ketones, disulfides, thiols, ketoacids, amines, amino acids, alcohols, alkenes, alkanes, and the like. In connection with the inventive methods, the term “target compound” does not include enzyme substrates that undergo a hydrolytic transformation that results in the creation or removal of an acidic or basic functionality, such as a carboxylic acid group. Target compounds are often chiral and/or transformed into chiral compounds by enzymes, and enrichment in single stereoisomers can occur.
- As used herein, the term “pH indicator” means any material or substance that changes its properties in response to a change in pH. Preferred changes in properties include a change in optical properties, such as a color change. Examples of pH indicators useful in the practice of the present invention include, but are not limited to, cresol red, m-cresol purple, bromothymol blue, bromophenol red, bromophenol blue, phenol red, and phenolphthalein. The pH indicator can be selected independently for each screen to determine the pH range or match a desired pH range for the enzyme to be detected. For example, m-cresol purple is yellow at a pH of about 7.4 and purple at a pH of about 9.0. Cresol red is yellow at a pH of about 7.2 and red at a pH of about 8.8. Bromothymol blue is yellow at a pH of about 6.0 and red at a pH of about 7.6. Bromophenol red is yellow at a pH of about 5.2 and red at a pH of about 6.8. Bromophenol blue is yellow at a pH of about 3.0 and blue at a pH of about 4.6. Phenol red is yellow at a pH of about 6.8 and red at a pH of about 8.2. Phenolphthalein is colorless at a pH of about 8.0 and pink at a pH of about 9.8. Other pH indicators can be selected depending on the desired pH range for the reaction and the desired color change.
- The conditions of the determination step can be adjusted to favor the detection or screening of an enzyme with a desired pH optimum by adjusting the pH of the reaction mixture used in the screen. For example, when an amino acid dehydrogenase that functions at pH 6 is sought, the reagent composition used for the screen can be buffered at a pH of 6 using a buffer that has its optimum buffering capacity near pH 6, and a pH indicator can be selected that changes color within the range of pH 5 to 7. Similarly, when an alcohol dehydrogenase that catalyzes the oxidation of a target alcohol at pH 9 is sought, the reagent composition used for the screen can be buffered at a pH of 9 using a buffer that has its optimum buffering capacity near pH 9, and a pH indicator can be selected that changes color within the range of pH 8 to 10. Typically, the pH indicator is selected such that it exhibits a color change in response to a change in pH within a range of about 1 to 1.5 pH units on either side of the desired pH for the reaction.
- If a buffer is used, the concentration of the buffer is preferably adjusted in order to maintain a desired initial pH for the screening reaction mixture and to reduce or eliminate small changes in pH not caused by the desired redox reaction. However, the concentration of the buffer should not be so high as to impede the change of pH that occurs as the reaction catalyzed by the oxidoreductase proceeds. The buffer may be any substance that helps maintain the desired initial pH of the solution. Examples include potassium phosphate, sodium phosphate, potassium borate, sodium borate, sodium acetate, potassium acetate, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, TRIS, PIPES, HEPES, MOPS, TEA, CHES, and the like. A listing of some useful biological buffers along with the pH ranges at which they are most effective as buffers can be found in theCatalog of Biochemicals and Reagents for Life Science Research; Sigma Chemical: St, Louis, 1998; p 1871. Often, desirable buffer concentrations must be determined experimentally. However, typical buffer concentrations using the method of the present invention are from about 0.01 mM to about 20 mM, and preferably from about 0.05 mM to about 5 mM.
- The above-described method can be used to determine activity of any enzyme that causes a pH change when it catalyzes the reaction of a target compound, and preferably is used to determine activity of oxidoreductases. As used herein, “oxidoreductase” refers to an enzyme capable of performing an oxidation reaction or reduction reaction. Nonlimiting examples of oxidoreductases include a reductase, an oxidase, a dehydrogenase, a ketoreductase, an alcohol dehydrogenase, a carbonyl reductase, an aldehyde dehydrogenase, an amino acid dehydrogenase, an amine oxidase, a disulfide reductase, an enoate reductase, and a mixed function oxidase. A listing of such enzymes can be found inEnzyme Nomenclature; Webb, E. C., Ed. Academic: Orlando 1984; pp 20-141, the disclosure of which is incorporated herein by reference.
- Often enzymes, and particularly oxidoreductases, require cofactors or cosubstrates for optimal activity. As used herein, the term “cofactor” means any molecule that participates in a chemical transformation of the target compound, including cofactors and cosubstrates. Nonlimiting examples of cofactors include nicotinamide cofactors, flavins, and derivatives and analogs thereof.
- As used herein, “nicotinamide cofactor” refers to any type of the oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD+ and NADH, respectively) and the oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate (NADP+ and NADPH, respectively) and derivatives and analogs thereof. With regard to a nicotinamide cofactor, the term “derivative” means any compound containing a pyridine structural element, including nicotinamides that have been chemically modified by attachment to soluble or insoluble polymeric materials. Some examples of derivatives of nicotinamide cofactors are described in U.S. Pat. No. 5,106,740, and Mansson and Mosbach Methods in Enzymology (1987) 136, 3-45, the disclosures of which are incorporated herein by reference. The term “analogs,” as used herein, refers to materials that undergo a formal hydride transfer in a redox reaction similar to that undergone by nicotinamide cofactors. Examples of analogs of nicotinamide cofactors useful in the practice of the present invention include compounds described in U.S. Pat. No. 5,801,006, the disclosure of which is incorporated herein by reference. Other suitable cofactors, as defined herein, can be used in the practice of the invention, as would be recognized by those skilled in the art.
- In the practice of the invention, the nicotinamide cofactors can be used in equimolar quantities relative to the target ketone, alcohol, amine or amino acid, or the cofactors may be recycled, if desired. Numerous methods for the recycling of nicotinamide cofactors are well-known in the art, and any of these methods may be used in the practice of the present invention. Some of the methods for recycling nicotinamide cofactors are described in G. L. Lemiere, et al., Tetrahedron Letters, 26, 4257 (1985); in “Enzymes as Catalysts for Organic Synthesis,” pp. 19-34, M. Schneider, Ed., Reidel Dordecht, 1986; in Z. Shaked and G. M. Whitesides, J. Am. Chem. Soc. 102, 7104-5 (1980); and J. B. Jones and T. Takamura, Can. J. Chem. 62, 77 (1984); the disclosures of which are incorporated herein by reference. In the use of these recycling methods, an amount of about 0.0001 mole to about 0.05 mole of nicotinamide cofactor is used per mole of ketone to be reduced or reductively aminated, per mole of 2-ketoacid to be reductively aminated, or per mole of alcohol or amine or amino acid to be oxidized, providing a recycle number for the cofactor of from about 20 to about 10,000.
-
- In the above scheme, and the schemes set forth below, A and B are independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocyclic, and the like. As shown in the balanced equation in Scheme 1, one proton is consumed in the reaction for each molecule of nicotinamide cofactor oxidized and each molecule of alcohol formed. As the reaction progresses, the consumption of protons causes the pH of the reaction mixture to rise. By including a suitable pH indicator in the reaction mixture, the presence of an alcohol dehydrogenase is indicated by a change in color of the reaction mixture. Although positive reactions can be detected spectrophotometrically, if desired, the use of a colorimetric pH indicator has the added advantage that the presence of oxidoreductase enzymes can be detected visually and without expensive instrumentation.
- Because of the reversibility of most reactions catalyzed by oxidoreductases, an oxidation reaction can also be used for screening and detection. For example, an alcohol dehydrogenase or carbonyl reductase catalyzes the oxidation of an alcohol to form an oxidized carbonyl compound, shown in Scheme 2.
- Thus, the presence of an oxidoreductase catalyzing this reaction can be detected using a reaction mixture containing an oxidized nicotinamide cofactor, an alcohol, and a pH indicator. In this case, the pH of the reaction mixture will decrease as the reaction progresses, and the decrease in pH is detected by the change in color of the pH indicator.
- As used herein, the term “carbonyl compound” means any chemical compound that has incorporated into it a functional group consisting of a carbon-oxygen double bond. The terms “carbonyl reductase”, “ketoreductase”, and “alcohol dehydrogenase” mean any enzyme that can catalyze the chemical reduction of a carbonyl group in the presence of a nicotinamide cofactor.
- With oxidoreductases that produce amines and amino acids by reductive amination, ammonia or ammonium ion is also a reactant. Thus, when screening for an amine or amino acid dehydrogenase using the method of the present invention, ammonia or a salt of ammonium ion is also included with a pH indicator, a reduced nicotinamide cofactor, and a ketone or ketoacid to be reductively aminated. The reaction is shown in Scheme 3.
- For detection of an amine or amino acid dehydrogenase that can oxidize an amine or amino acid to the corresponding ketone or ketoacid using then method of the present invention, the reaction mixture for detection contains a pH indicator, an oxidized nicotinamide cofactor, and an amine or amino acid to be oxidized. The reaction for the oxidation of an amine or amino acid using an amine or amino acid dehydrogenase is depicted in Scheme 4:
- The above-described method can be used to detect enzymes using a second reaction that can be coupled to the enzyme-catalyzed reaction to be screened. For example, in screening for an aminotransferase that catalyzes the transamination of a ketone or ketoacid with L-aspartic acid as the donor, the reaction products are an amine or amino acid and oxaloacetate. In a second reaction, the oxaloacetate can be decarboxylated to pyruvate, with the consumption of a proton. Thus, aminotransferase activity can be detected by detecting an increase in the pH of the reaction mixture because the decarboxylation of oxaloacetate is coupled to the transamination reaction to be screened.
- This method is particularly useful for screening for enzymes to perform specific chemical transformations of target compounds that are intermediates in chemical syntheses. Thus, after an enzyme has been determined to have activity for a particular target compound, it can be used to convert that target compound to a useful chemical intermediate, as described above. Useful chemical intermediates include alcohols, amines, alpha-amino acids, beta-amino acids, gamma-amino acids, aldehydes, ketones, carboxylic acids, esters, amides, and the like.
- As discussed above, enzymes often require cofactors or cosubstrates for optimal activity. Accordingly, when converting a target compound, preferably a cofactor is present with the enzyme. Suitable cofactors are set forth above.
- In the pharmaceutical industry, it is often desirable to chemically transform target compounds into one stereoisomer to the substantial exclusion of another. More specifically, it is desirable to obtain these compounds in more than about 90% enantiomeric excess (ee), preferably in about 95% ee, and still more preferably in about 98% ee, because of the considerable difficulty and the tremendous waste of material in separating enantiomeric products from a racemic mixture. Because enzymes can perform chemical transformations exclusively forming one enantiomeric product and often are easier to use and more cost-effective than performing an asymmetric synthesis, new enzymes that can act upon target compounds are sought after, such as a carbonyl reductase that produces of a single stereoisomer of a alcohol in 98% ee.
- In a particularly preferred embodiment, the target compound is a ketone that is not a ketoacid, and the target compound is converted to an amine, preferably a chiral amine, in the presence of an amine dehydrogenase. Preferably the above-described screening method is used to first determine enzymatically-active amino acid dehydrogenases. Further screening is then performed on the enzymatically-active amino acid dehydrogenases, again, in accordance with the procedures described above, to identify enzymatically-active amine dehydrogenases. The thus identified enzymatically-active amine dehydrogenase is then provided in a solution together with a ketone, ammonia and reduced nicotinamide cofactor to synthesize an amine.
- The invention is now further described by the following examples, which are given here for illustrative purposes only and are not intended to limit the scope of the invention.
- A gene encoding the alcohol dehydrogenase YPR1 (described by Nakamura, K., et al.,Bioscience, Biotechnology and Biochemistry, (1997) 61, 375-377), is subjected to mutagenesis by error-prone PCR according to the method of May, O., et al., (Nature Biotechnology, (2000) 18, 317-320). The error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as a template dissolved in PCR buffer (10 mM TRIS, 1.5 mM MgCl2, 50 mM KCl, pH 8.3), and also containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase (Roche Diagnostics, Indianapolis, Ind.). Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C. The PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector (Invitrogen, Carlsbad, Calif.) which has been digested with the same restriction enzymes. The resulting YPR 1 mutant library is transformed into the E. coli host strain LMG 194 (Invitrogen, Carlsbad, Calif.) and plated on LB agar supplied with 100 μg/mL ampicillin. Individual transformants are inoculated into 96-well microtiter plates (hereafter referred to as master plates) containing 0.2 mL LB Broth with 100 μg/mL ampicillin, and growth is allowed to take place for 8 to 16 hours at 37° C. with shaking at 200 rpm. Each well in each master plate is then re-inoculated by a replica plating technique into a new second stage 96-well plate pre-loaded with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5-10 hours at 37° C. with shaking at 200 rpm. The second stage plates are then centrifuged at 14,000 rpm for 20 minutes, and the supernatant is decanted. The cell pellet in each well is washed with 200 mL of water. The washed cell pellet is suspended in 30 mL of B-Per Bacterial Protein Extraction Reagent (Pierce Chemical Co., Rockford, Ill.), hereinafter “B-Per.” After mixing, the suspension of cells in B-Per reagent is allowed to stand for 10 minutes at room temperature, and a reaction solution having the following composition is then added to each well in the plate:
- 7.5 μL of a pH 6.5 solution containing 8 μg/mL of NADPH
- 7.5 μL of a pH 6.5 50% DMSO solution containing 0.25 M ethyl-4-chloro-3-ketobutyrate
- 155 μL of 1 mM potassium phosphate buffer, pH 6.5
- 1.5 μL of a 4 μg/mL solution of cresol red indicator
- Wells containing an alcohol dehydrogenase that catalyzes the reduction of the target compound ethyl-4-chloro-3-ketobutyrate can be identified easily as their color changes from an initial yellow to an orange or red color. These wells are correlated to the original wells in the master plates to obtain the original clones of mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- The procedure of Example 1 is repeated, replacing the ethyl-4-chloro-3-ketobutyrate with ethyl-3-phenyl-3-ketopropionate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- The procedure of Example 1 is repeated, replacing the ethyl-4-chloro-3-ketobutyrate with ethyl-indan-2-one-1-carboxylate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- The procedure of Example 1 is repeated, replacing the ethyl-4-chloro-3-ketobutyrate with ethyl-4-phenyl-4-ketobutyrate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- The procedure of Example 1 is repeated, replacing the reaction solution with a reaction solution of the following composition:
- 7.5 μL of a pH 6.5 solution containing 8 μg/mL of NADPH
- 7.5 μL of pH 6.5 DMSO solution containing 0.25 M ethyl-3-phenyl-3-ketopropionate
- 155 μL of a 1 mM potassium phosphate buffer, pH 7.0
- 1.5 μL of a 4 μg/mL solution of a cresol red indicator
- Wells containing an alcohol dehydrogenase that reduces ethyl-3-phenyl-3-ketopropionate can be identified easily as the color changes from an initial yellow to a red color. At least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction is identified.
- The procedure of Example 5 is repeated, replacing the ethyl-3-phenyl-3-ketopropionate with ethyl-indan-2-one-1-carboxylate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- The procedure of Example 5 is repeated, replacing the ethyl-3-phenyl-3-ketopropionate with ethyl-4-phenyl-4-ketobutyrate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- The procedure of Example 5 is repeated, replacing the ethyl-3-phenyl-3-ketoproponiate with ethyl-cyclohexanone-2-carboxylate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- The procedure of Example 1 is repeated, replacing the reaction solution with a reaction solution of the following composition:
- 7.5 μL of a pH 6.5 solution containing 8 μg/mL of NADPH
- 7.5 μL of a pH 6.5 50% DMSO solution containing 0.25 M ethyl-2-ethyl-3-ketobutyrate
- 155 μL of a 2 mM potassium phosphate buffer, pH 6.5
- 1.5 μL of a 4 μg/mL solution of bromothymol blue indicator
- Wells containing an alcohol dehydrogenase that reduces ethyl-2-ethyl-3-ketobutyrate can be identified easily as the color changes from an initial yellow to a blue color.
- The procedure of Example 9 is repeated, replacing the ethyl-2-ethyl-3-ketobutyrate with ethyl-2-allyl-3-ketobutyrate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- The procedure of Example 9 is repeated, replacing the ethyl-2-ethyl-3-ketobutyrate with ethyl-2-phenyl-3-ketobutyrate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- The procedure of Example 9 is repeated, replacing the ethyl-2-ethyl-3-ketobutyrate with ethyl-2-benzyl-3-ketobutyrate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired reduction reaction.
- A gene encoding the alcohol dehydrogenase Alr1 (Yamada, et al.,FEMS Microbiology Letters, (1990) 70, 45-48) is subjected to mutagenesis by error-prone PCR according to the method of May et al. The error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PCR buffer containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase. Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C. The PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector which has been digested with the same restriction enzymes. The resulting Alr1 mutant library is transformed into an E. coli host strain LMG194 and plated on LB agar supplied with 100 μg/mL ampicillin. Individual transformants are inoculated into master plates containing 0.2 mL LB Broth with 100 μg/mL ampicillin, and growth is allowed to take place for 8-16 hours at 37° C. with shaking at 200 rpm. Each well in each master plate is then re-inoculated by a replica plating technique into a new second stage 96-well plate pre-loaded with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5 to 10 hours at 37° C. with shaking at 200 rpm. The second stage plates are then centrifuged at 14,000 rpm for 20 minutes, and the supernatant is decanted. The cell pellet in each well is washed with 200 mL of water. The washed cell pellet is suspended in 30 mL of B-Per. After mixing, the suspension of cells in B-Per reagent is allowed to stand for 10 minutes at room temperature, and a solution having the following composition is then added to each well in the plate:
- 7.5 μL of a pH 8.0 solution containing 20 μg/ml of NADP+
- 7.5 μL of a pH 8.0 50% DMSO solution containing 0.25 M (2R,3S)-ethyl-2-ethyl-3-hydroxybutyrate
- 155 μL of 2 mM potassium phosphate buffer, pH 8.0
- 1.5 μL of a 4 μg/ml solution of bromothymol blue indicator
- Wells in which the color changes from an initial blue to a yellow color contain mutant alcohol dehydrogenases that catalyze the oxidation of the target alcohol (2R,3S)-ethyl-2-ethyl-3-hydroxybutyrate. These wells are correlated to the original wells in the master plates to obtain the original clones of mutant alcohol dehydrogenases catalyzing the desired oxidation reaction.
- A gene encoding leucine dehydrogenase fromB. stearothermophilus (Nagata, et al. Biochemistry (1998) 27, 9056) is subjected to mutagenesis by error-prone PCR according to the method of May et al. The error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PCR buffer also containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase. Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C. The PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector which has been digested with the same restriction enzymes. The resulting leucine dehydrogenase mutant library is transformed into an E. coli host strain LMG194 and plated on LB agar supplied with 100 μg/mL ampicillin. Individual transformants are inoculated into master plates containing 0.2 mL LB Broth with 100 μg/mL ampicillin, and growth is allowed to take place for 8 to 16 hours at 37° C. with shaking at 200 rpm. Each well in each master plate is then re-inoculated by a replica plating technique into a new second stage 96-well plate pre-loaded with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5-10 hours at 37° C. with shaking at 200 rpm. The second stage plates are then centrifuged at 14,000 rpm for 20 minutes, and the supernatant is decanted. The cell pellet in each well is washed with 200 mL of water. The washed cell pellet is suspended in 30 mL of B-Per After mixing, the suspension of cells in B-Per reagent is allowed to stand for 10 minutes at room temperature, and a solution having the following composition is then added to each well in the plate:
- 7.5 μL of a pH 6.5 solution containing 8 μg/mL of NADH
- 7.5 μL of a pH 6.5 50% DMSO solution containing 0.25 M 3,3-dimethyl-2-ketobutyrate
- 155 μL f 1 mM potassium phosphate buffer, pH 6.5, containing 100 mM ammonium chloride
- 1.5 μL of a 4 μg/mL solution of cresol red indicator
- Wells in which the color changes from an initial yellow to an orange or red color contain leucine dehydrogenase that catalyzes the reductive amination of the target 2-ketoacid 3,3-dimethyl-2-ketobutyrate. These wells are correlated to the original wells in the master plates to obtain the original clones of mutant leucine dehydrogenase that catalyzes the desired reductive amination reaction.
- The procedure of Example 14 is repeated, replacing 3,3 -dimethyl-2-ketobutyrate with 4-(methylphosphinyl)-2-ketobutyrate, thereby identifying at least one mutant dehydrogenase that catalyzes the desired reductive amination reaction.
- The procedure of Example 14 is repeated, replacing 3,3-dimethyl-2-ketobutyrate with 3-(2-naphthyl)pyruvate, thereby identifying at least one mutant dehydrogenase that catalyzes the desired reductive amination reaction.
- The procedure of Example 14 is repeated, replacing the 3,3-dimethyl-2-ketobutyrate with 3-(1-naphthyl)pyruvate, thereby identifying at least one mutant dehydrogenase that catalyzes the desired reductive amination reaction.
- The procedure of Example 14 is repeated, replacing 3,3-dimethyl-2-ketobutyrate with 4-phenyl-2-ketobutyrate, thereby identifying at least one mutant dehydrogenase that catalyzes the desired reductive amination reaction.
- The procedure of Example 14 is repeated replacing the 3,3-dimethyl-2-ketobutyrate with 4,4-dimethyl-2-ketopentanoate, thereby identifying at least one mutant dehydrogenase that catalyzes the desired reduction reaction.
- A gene encoding the leucine dehydrogenase fromB. stearothermophilus is subjected to mutagenesis by error-prone PCR according to the method of May et al. The error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PCR buffer (10 mM TRIS, 1.5 mM MgCl2, 50 mM KCl, pH 8.3), and also containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase. Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C. The PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector which has been digested with the same restriction enzymes. The resulting leucine dehydrogenase mutant library is transformed into an E. coli host strain LMG194 and plated on LB agar supplied with 100 μg/mL ampicillin. Individual transformants are inoculated into 96-well master plates containing 0.2 mL LB Broth with 100 μg/mL ampicillin, and growth is allowed to take place for 8-16 hours at 37° C. with shaking at 200 rpm. Each well in each master plate is then re-inoculated by a replica plating technique into a new second stage 96-well plate pre-loaded with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5-10 hours at 37° C. with shaking at 200 rpm. The second stage plates are then centrifuged at 14,000 rpm for 20 minutes, and the supernatant is decanted. The cell pellet in each well is washed with 200 mL of water. The washed cell pellet is suspended in 30 mL of B-Per. After mixing, the suspension of cells in B-Per reagent is allowed to stand for 10 minutes at room temperature, and a solution having the following composition is then added to each well in the plate:
- 7.5 μL of a pH 8.0 solution containing 20 μg/ml of NAD+
- 7.5 μL of a pH 8.0 50% DMSO solution containing 0.25 M L-tert-leucine (S-3,3-dimethyl-2-aminobutyrate)
- 155 μL of 2 mM potassium phosphate buffer, pH 8.0, containing 100 mM ammonium chloride
- 1.5 μL of a 4 μg/ml solution of bromothymol blue indicator
- Wells in which the color changes from an initial blue to a yellow color contain mutant leucine dehydrogenases that catalyze the oxidation of the target amino acid. These wells are correlated to the original wells in the master plates to obtain the original clones of mutant leucine dehydrogenases catalyzing the desired oxidation reaction.
- The procedure of Example 20 is repeated replacing the L-tert-leucine with S-phosphinothricin, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired oxidation reaction.
- The procedure of Example 20 is repeated, replacing the L-tert-leucine with S-(2-naphthyl)alanine, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired oxidation reaction.
- The procedure of Example 20 is repeated, replacing the L-tert-leucine with D-tert-leucine, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired oxidation reaction.
- The procedure of Example 20 is repeated, replacing L-tert-leucine with S-4-phenyl-2-aminobutyrate, thereby identifying at least one mutant alcohol dehydrogenase that catalyzes the desired oxidation reaction.
- The procedure of Example 20 is repeated, replacing the L-tert-leucine with D-tyrosine, thereby identifying at lest one mutant alcohol dehydrogenase that catalyzes the desired oxidation reaction.
- A gene encoding leucine dehydrogenase fromB. stearothermophilus is subjected to mutagenesis by error-prone PCR according to the method of May, et al. The error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PCR buffer containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase. Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C. The PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector which has been digested with the same restriction enzymes. The resulting leucine dehydrogenase mutant library is transformed into an E. coli host strain LMG194 and plated on LB agar supplied with 100 μg/mL ampicillin. Individual transformants are inoculated into master plates containing 0.2 mL LB Broth with 100 μg/mL ampicillin, and growth is allowed to take place for 8-16 hours at 37° C. with shaking at 200 rpm. Each well in each master plate is then re-inoculated by a replica plating technique into a new second stage 96-well plate pre-loaded with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5-10 hours at 37° C. with shaking at 200 rpm. The second stage plates are then centrifuged at 14,000 rpm for 20 minutes, and the supernatant is decanted. The cell pellet in each well is washed with 200 mL of water. The washed cell pellet is suspended in 30 mL of B-Per. After mixing, the suspension of cells in B-Per reagent is allowed to stand for 10 minutes at room temperature, and a solution having the following composition is then added to each well in the plate:
- 7.5 μL of a pH 6.5 solution containing 8 μg/mL of NADH
- 7.5 μL of a pH 6.5 50% DMSO solution containing 0.25 M acetophenone
- 155 μL of 1 mM potassium phosphate buffer, pH 6.5, containing 100 mM ammonium chloride
- 1.5 μL of a 4 μg/mL solution of cresol red indicator
- Wells in which the color change from an initial yellow to an orange or red color contain leucine dehydrogenases that catalyzes the reductive amination of the target ketone acetophenone. These wells are correlated to the original wells in the master plates to obtain the original clones of mutant leucine dehydrogenase that catalyzes the desired reductive amination reaction.
- A gene encoding the leucine dehydrogenase fromB. stearothermophilus is subjected to mutagenesis by error-prone PCR according to the method of May, et al. The error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PCR buffer containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase. Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C. The PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector which has been digested with the same restriction enzymes. The resulting leucine dehydrogenase mutant library is transformed into an E. coli host strain LMG1 94 and plated on LB agar supplied with 100 μg/mL ampicillin. Individual transformants are inoculated into master plates containing 0.2 mL LB Broth with 100 μg/mL ampicillin, and growth is allowed to take place for 8-16 hours at 37° C. with shaking at 200 rpm. Each well in each master plate is then re-inoculated by a replica plating technique into a new second stage plate pre-loaded with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5 to 10 hours at 37° C. with shaking at 200 rpm. The second stage plates are then centrifuged at 14,000 rpm for 20 minutes, and the supernatant is decanted. The cell pellet in each well is washed with 200 mL of water. The washed cell pellet is suspended in 30 mL of B-Per Bacterial Protein Extraction Reagent. After mixing, the suspension of cells in B-Per reagent is allowed to stand for 10 minutes at room temperature, and a solution having the following composition is then added to each well in the plate:
- 7.5 μL of a pH 8.0 solution containing 20 μg/mL of NAD+
- 7.5 μL of a pH 8.0 50% DMSO solution containing 0.25 M S-1-phenylethylamine
- 155 μL of 2 mM potassium phosphate buffer, pH 8.0, containing 100 mM ammonium chloride
- 1.5 μL of a 4 μg/mL solution of bromothymol blue indicator
- Wells in which the color changes from blue initially to yellow contain mutant leucine dehydrogenase that catalyze the oxidation of the target amine. These wells are correlated to the original wells in the master plates to obtain the original clones of mutant leucine dehydrogenases catalyzing the desired oxidation reaction.
- A gene encoding the leucine dehydrogenase fromB. stearothermophilus is subjected to mutagenesis by error-prone PCR according to the method of May, et al. The error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PCR buffer containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase. Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C. The PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector which has been digested with the same restriction enzymes. The resulting leucine dehydrogenase mutant library is transformed into an E. coli host strain LMG194 and plated on LB agar supplied with 100 μg/mL ampicillin. Individual transformants are inoculated into master plates containing 0.2 mL LB Broth with 100 μg/mL ampicillin, and growth is allowed to take place for 8-16 hours at 37° C. with shaking at 200 rpm. Each well in each master plate is then re-inoculated by a replica plating technique into a new second stage plate pre-loaded with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5-10 hours at 37° C. with shaking at 200 rpm. The second stage plates are then centrifuged at 14,000 rpm for 20 minutes, and the supernatant is decanted. The cell pellet in each well is washed with 200 mL of water. The washed cell pellet is suspended in 30 mL of B-Per. After mixing, the suspension of cells in B-Per reagent is allowed to stand for 10 minutes at room temperature, and a solution having the following composition is then added to each well in the plate:
- 7.5 μL of a pH 8.0 solution containing 20 μg/mL of NAD+
- 7.5 μL of a pH 8.0 50% DMSO solution containing 0.25 M R-1-phenylethylamine
- 155 μL of 2 mM potassium phosphate buffer, pH 8.0, containing 100 mM ammonium chloride
- 1.5 μL of a 4 μg/mL solution of bromothymol blue indicator
- Wells in which the color changes from blue initially to yellow contain mutant leucine dehydrogenase that catalyze the oxidation of the target amine. These wells are correlated to the original wells in the master plates to obtain the original clones of mutant leucine dehydrogenases catalyzing the desired oxidation reaction.
- One hundred units of an amine dehydrogenase generated by mutagenesis and screening of leucine dehydrogenase as described in any one of Examples 26 to 28 above is incubated at 45° C. in 100 milliliters of a solution maintained at pH 6.5 containing potassium phosphate (1 millimole), NADH (0.01 millimole), ammonium formate (25 millimoles), and formate dehydrogenase fromCandida boidinii (100 units). Acetophenone (10 millimoles) is added slowly over one hour with stirring, and the reaction is allowed to proceed for an additional 4 hours. After basification of the reaction mixture to pH 12 and extraction with methyl t-butyl ether, analysis of the reaction products is carried out by gas chromatography to determine the yield of 1-phenylethylamine. Chiral analysis is carried out by chiral gas chrmoatography using a ChiraDex CB column (Advanced Separation Technology, Whippany, N.J. USA).
- The method of Example 29 is carried out except that the amine dehydrogenase is an R-1-phenylethylamine dehydrogenase and the product is R-1-phenylethylamine.
- The method of Example 29 is carried out except that the amine dehydrogenase is an S-1-phenylethylamine dehydrogenase and the product is S-1-phenylethylamine.
- The method of Example 30 is carried out except that acetophenone is replaced by p-chloroacetophenone and the product is R-1-(p-chlorophenyl)ethylamine.
- The method of Example 31 is carried out except that acetophenone is replaced by p-chloroacetophenone and the product is S-1-(p-chlorophenyl)ethylamine.
- The method of Example 30 is carried out except that acetophenone is replaced by m-bromoacetophenone and the product is R-1-(m-bromophenyl)ethylamine.
- The method of Example 31 is carried out except that acetophenone is replaced by m-bromoacetophenone and the product is S-1-(m-bromophenyl)ethylamine.
- One hundred units of an alcohol dehydrogenase, generated by mutagenesis and screening of the alr1 gene as described in Example 13 above, is incubated at 45 EC in 100 milliliters of a solution maintained at pH 6.5 containing potassium phosphate (1 millimole), NADPH (0.01 millimole), sodium formate (25 millimoles), and a NADP-utilizing formate dehydrogenase P3 (obtained from Juelich Fine Chemcials, Juelich, Germany; catalog number 25.10; 100 units). Acetophenone (10 millimoles) is added slowly over one hour with stirring, and the reaction is allowed to proceed for an additional 4 hours. The reaction mixture is extracted with methyl t-butyl ether, and analysis of the reaction products is carried out by gas chromatography to determine the yield of 1-phenylethanol. Chiral analysis is carried out by chiral gas chromatography using a ChiraDex CB column (Advanced Separation Technology, Whippany, N.J. USA).
- The method of Example 36 is carried out except that the alcohol dehydrogenase is determined to be an R-1-phenylethanol dehydrogenase and the product is R-1-phenylethanol.
- The method of Example 36 is carried out except that the alcohol dehydrogenase is determined to be an 5-1-phenylethanol dehydrogenase and the product is S-1-phenylethanol,
- The method of Example 36 is carried out except that acetophenone is replaced by p-chloroacetophenone, the alcohol dehydrogenase is determined to be an R-1-(p-chlorophenyl)ethanol dehydrogenase and the product is R-1-(p-chlorophenyl)ethanol.
- The method of Example 36 is carried out except that acetophenone is replaced by p-chloroacetophenone, the alcohol dehydrogenase is determined to be an S-1-(p-chlorophenyl)ethanol dehydrogenase and the product is S-1-(p-chlorophenyl)ethanol.
- The method of Example 36 is carried out except that acetophenone is replaced by m-bromoacetophenone, the alcohol dehydrogenase is determined to be an R-1-(m-bromophenyl)ethanol dehydrogenase, and the product is R-1-(m-bromophenyl)ethanol.
- The method of Example 36 is carried out except that acetophenone is replaced by m-bromoacetophenone, the alcohol dehydrogenase is determined to be an S-1-(m-bromophenyl)ethanol dehydrogenase, and the product is S-1-(m-bromophenyl)ethanol.
- The preceding description has been presented with reference to presently preferred embodiments of the invention. Workers skilled in the art and technology to which this invention pertains will appreciate that alterations and changes in the described methods and kits may be practiced without meaningfully departing from the spirit and scope of this invention. Accordingly, the foregoing description should not be read as pertaining only to the precise methods and kits described, but rather should be read consistent with and as support to the following claims, which are to have their fullest and fair scope.
Claims (30)
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/039,952 US7202070B2 (en) | 2000-10-31 | 2001-10-24 | Method for reductive amination of a ketone using a mutated enzyme |
AU2002232603A AU2002232603A1 (en) | 2000-10-31 | 2001-10-30 | Method for chemical transformation using a mutated enzyme |
PCT/US2001/048577 WO2002036742A2 (en) | 2000-10-31 | 2001-10-30 | Method for chemical transformation using a mutated enzyme |
US11/786,054 US7642073B2 (en) | 2000-10-31 | 2007-04-09 | Method for reductive amination of a ketone using a mutated enzyme |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US70242100A | 2000-10-31 | 2000-10-31 | |
US28837801P | 2001-05-03 | 2001-05-03 | |
US10/039,952 US7202070B2 (en) | 2000-10-31 | 2001-10-24 | Method for reductive amination of a ketone using a mutated enzyme |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US70242100A Continuation-In-Part | 2000-10-31 | 2000-10-31 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/786,054 Continuation US7642073B2 (en) | 2000-10-31 | 2007-04-09 | Method for reductive amination of a ketone using a mutated enzyme |
Publications (2)
Publication Number | Publication Date |
---|---|
US20020061564A1 true US20020061564A1 (en) | 2002-05-23 |
US7202070B2 US7202070B2 (en) | 2007-04-10 |
Family
ID=27365635
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/039,952 Expired - Lifetime US7202070B2 (en) | 2000-10-31 | 2001-10-24 | Method for reductive amination of a ketone using a mutated enzyme |
US11/786,054 Expired - Lifetime US7642073B2 (en) | 2000-10-31 | 2007-04-09 | Method for reductive amination of a ketone using a mutated enzyme |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/786,054 Expired - Lifetime US7642073B2 (en) | 2000-10-31 | 2007-04-09 | Method for reductive amination of a ketone using a mutated enzyme |
Country Status (3)
Country | Link |
---|---|
US (2) | US7202070B2 (en) |
AU (1) | AU2002232603A1 (en) |
WO (1) | WO2002036742A2 (en) |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080248539A1 (en) * | 2006-10-02 | 2008-10-09 | Codexis, Inc. | Compositions and methods for producing stereoisomerically pure statins and synthetic intermediates therefor |
US20080318295A1 (en) * | 2007-02-08 | 2008-12-25 | Codexis, Inc. | Ketoreductases and Uses Thereof |
US20090093031A1 (en) * | 2007-08-24 | 2009-04-09 | Codexis, Inc. | Ketoreductase Polypeptides for the Production of (R)-3-Hydroxythiolane |
US20090155863A1 (en) * | 2007-09-28 | 2009-06-18 | Codexis, Inc. | Ketoreductase polypeptides and uses thereof |
US20090162909A1 (en) * | 2007-10-01 | 2009-06-25 | Codexis, Inc. | Ketoreductase Polypeptides for the Production of Azetidinone |
US20100151534A1 (en) * | 2008-08-27 | 2010-06-17 | Codexis, Inc. | Ketoreductase polypeptides for the production of a 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine |
US20100173369A1 (en) * | 2008-08-27 | 2010-07-08 | Codexis, Inc. | Ketoreductase polypeptides for the production of 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine |
US8273554B2 (en) | 2008-08-29 | 2012-09-25 | Codexis, Inc. | Ketoreductase polypeptides for the stereoselective production of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one |
US8512973B2 (en) | 2007-09-13 | 2013-08-20 | Codexis, Inc. | Methods of using engineered ketoreductase polypeptides for the stereoselective reduction of acetophenones |
US9040262B2 (en) | 2010-05-04 | 2015-05-26 | Codexis, Inc. | Biocatalysts for ezetimibe synthesis |
US9102959B2 (en) | 2009-08-19 | 2015-08-11 | Codexis, Inc. | Ketoreductase polypeptides for the preparation of phenylephrine |
CN112394050A (en) * | 2019-08-19 | 2021-02-23 | 中国科学院天津工业生物技术研究所 | Detection method for high-throughput screening of ketone compounds and application of detection method in enzyme screening |
Families Citing this family (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7202070B2 (en) * | 2000-10-31 | 2007-04-10 | Biocatalytics, Inc. | Method for reductive amination of a ketone using a mutated enzyme |
CN102482648B (en) * | 2009-06-22 | 2014-12-10 | 科德克希思公司 | Ketoreductase-mediated stereoselective route to alpha chloroalcohols |
US8921079B2 (en) | 2009-06-22 | 2014-12-30 | Codexis, Inc. | Transaminase reactions |
EP2847214B1 (en) | 2012-05-11 | 2017-12-27 | Codexis, Inc. | Engineered imine reductases and methods for the reductive amination of ketone and amine compounds |
DE102012017026A1 (en) | 2012-08-28 | 2014-03-06 | Forschungszentrum Jülich GmbH | Sensor for NADP (H) and development of alcohol dehydrogenases |
CN104513839B (en) * | 2013-09-30 | 2017-12-26 | 中国科学院天津工业生物技术研究所 | A kind of biocatalysis preparation method of D Terleus |
EP2963121A1 (en) * | 2014-07-03 | 2016-01-06 | Basf Se | Redox self-sufficient biocatalytic amination of alcohols |
CN106906190B (en) * | 2015-12-23 | 2019-10-18 | 中国科学院微生物研究所 | One group of leucine dehydrogenase and its encoding gene and application |
BR112018067523A8 (en) | 2016-03-02 | 2023-01-31 | Agrimetis Llc | METHODS FOR PREPARING L-GLUFOSINATE |
WO2019084950A1 (en) * | 2017-11-06 | 2019-05-09 | 凯莱英生命科学技术(天津)有限公司 | Transaminase mutant and use thereof |
CN109609474B (en) | 2018-12-28 | 2020-07-28 | 浙江工业大学 | Amino acid dehydrogenase mutant and application thereof in synthesis of L-glufosinate-ammonium |
CN111593077B (en) * | 2019-12-30 | 2021-10-01 | 南京朗恩生物科技有限公司 | Method for preparing (R) -4-chloro-3-hydroxy ethyl butyrate through biocatalysis |
CN113583988B (en) * | 2020-04-30 | 2023-09-12 | 沈阳药科大学 | Amino acid dehydrogenase mutant and application thereof |
CN112481230B (en) * | 2020-12-04 | 2021-12-07 | 浙江科技学院 | Omega-transaminase mutant obtained by DNA synthesis shuffling and combined mutation and application thereof |
Citations (23)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4518692A (en) * | 1983-09-01 | 1985-05-21 | Genetics Institute, Inc. | Production of L-amino acids by transamination |
US4710467A (en) * | 1983-07-29 | 1987-12-01 | Purification Engineering, Inc. | Process for preparing phenylalanine |
US4745059A (en) * | 1984-06-29 | 1988-05-17 | Hoechst Aktiengesellschaft | Process for the preparation of L-phenylalanine |
US5106740A (en) * | 1987-08-28 | 1992-04-21 | Hoechst Aktiengesellschaft | Immobilization of an isethiacyanate of a cofactor on a polymer |
US5316943A (en) * | 1988-06-14 | 1994-05-31 | Kidman Gene E | Racemic conversion of using a transaminase |
US5605793A (en) * | 1994-02-17 | 1997-02-25 | Affymax Technologies N.V. | Methods for in vitro recombination |
US5753470A (en) * | 1986-06-04 | 1998-05-19 | Hoechst Aktiengesellschaft | Process for preparing L-tertiary-Leucine and L-phosphinothricine by transamination |
US5798234A (en) * | 1993-10-18 | 1998-08-25 | Degussa Aktiengesellschaft | Method for the directed modification of enzymes, modified enzymes and their use |
US5801006A (en) * | 1997-02-04 | 1998-09-01 | Specialty Assays, Inc. | Use of NADPH and NADH analogs in the measurement of enzyme activities and metabolites |
US5837458A (en) * | 1994-02-17 | 1998-11-17 | Maxygen, Inc. | Methods and compositions for cellular and metabolic engineering |
US5854035A (en) * | 1996-02-22 | 1998-12-29 | Degussa Ag | Enzyme with leuDH activity, nucleotide sequence coding therefor and process for the prepartion of the enzyme |
US5958715A (en) * | 1995-06-26 | 1999-09-28 | Brf International | Method for quantitative measurement of an enzyme linked immunosorbent assay |
US5958672A (en) * | 1995-07-18 | 1999-09-28 | Diversa Corporation | Protein activity screening of clones having DNA from uncultivated microorganisms |
US5965408A (en) * | 1996-07-09 | 1999-10-12 | Diversa Corporation | Method of DNA reassembly by interrupting synthesis |
US6001574A (en) * | 1996-06-18 | 1999-12-14 | Diversa Corporation | Production and use of normalized DNA libraries |
US6030779A (en) * | 1995-07-18 | 2000-02-29 | Diversa Corporation | Screening for novel bioactivities |
US6054267A (en) * | 1995-12-07 | 2000-04-25 | Diversa Corporation | Method for screening for enzyme activity |
US6117679A (en) * | 1994-02-17 | 2000-09-12 | Maxygen, Inc. | Methods for generating polynucleotides having desired characteristics by iterative selection and recombination |
US6255092B1 (en) * | 1993-09-24 | 2001-07-03 | Daicel Chemical Industries Ltd. | Stereospecific alcohol dehydrogenase isolated from Candida parapsilosis, amino acid and DNA sequences therefor, and method of preparation thereof |
US6365380B2 (en) * | 2000-02-23 | 2002-04-02 | Pcbu Services, Inc. | Method for stereoselectively inverting a chiral center of a chemical compound using an enzyme and a metal catalyst |
US20020192786A1 (en) * | 1997-04-23 | 2002-12-19 | Kaneka Corporation | Producing optically active amino compounds |
US20030138930A1 (en) * | 2000-11-14 | 2003-07-24 | Applera Corporation | Isolated human dehydrogenaseproteins, nucleic acid molecules encoding these human dehydrogenase proteins, and uses thereof |
US6727083B2 (en) * | 1999-03-19 | 2004-04-27 | Sumitomo Chemical Company, Limited | Protein capable of catalyzing transamination stereoselectively, gene encoding said protein and use thereof |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5300437A (en) * | 1989-06-22 | 1994-04-05 | Celgene Corporation | Enantiomeric enrichment and stereoselective synthesis of chiral amines |
US5942644A (en) * | 1997-05-27 | 1999-08-24 | Biocatalytics, Inc. | Precursors for the production of chiral vicinal aminoalcohols |
US5916786A (en) * | 1997-12-19 | 1999-06-29 | Biocatalytics, Inc. | Method for the production of chiral 1,3-aminoalcohols |
US6207862B1 (en) * | 1997-12-19 | 2001-03-27 | Biocatalytics, Inc. | Precursors for the production of chiral 1,3-aminoalcohols |
DE19812004A1 (en) * | 1998-03-19 | 1999-09-30 | Forschungszentrum Juelich Gmbh | Dehydrogenases with improved NAD dependence, their production and use |
DE69940978D1 (en) * | 1998-11-10 | 2009-07-23 | Univ Toronto | CHEMICALLY MODIFIED MUTANT SERINHYDROLASES |
US7202070B2 (en) * | 2000-10-31 | 2007-04-10 | Biocatalytics, Inc. | Method for reductive amination of a ketone using a mutated enzyme |
-
2001
- 2001-10-24 US US10/039,952 patent/US7202070B2/en not_active Expired - Lifetime
- 2001-10-30 AU AU2002232603A patent/AU2002232603A1/en not_active Abandoned
- 2001-10-30 WO PCT/US2001/048577 patent/WO2002036742A2/en active Application Filing
-
2007
- 2007-04-09 US US11/786,054 patent/US7642073B2/en not_active Expired - Lifetime
Patent Citations (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4710467A (en) * | 1983-07-29 | 1987-12-01 | Purification Engineering, Inc. | Process for preparing phenylalanine |
US4518692A (en) * | 1983-09-01 | 1985-05-21 | Genetics Institute, Inc. | Production of L-amino acids by transamination |
US4745059A (en) * | 1984-06-29 | 1988-05-17 | Hoechst Aktiengesellschaft | Process for the preparation of L-phenylalanine |
US5753470A (en) * | 1986-06-04 | 1998-05-19 | Hoechst Aktiengesellschaft | Process for preparing L-tertiary-Leucine and L-phosphinothricine by transamination |
US5106740A (en) * | 1987-08-28 | 1992-04-21 | Hoechst Aktiengesellschaft | Immobilization of an isethiacyanate of a cofactor on a polymer |
US5316943A (en) * | 1988-06-14 | 1994-05-31 | Kidman Gene E | Racemic conversion of using a transaminase |
US6255092B1 (en) * | 1993-09-24 | 2001-07-03 | Daicel Chemical Industries Ltd. | Stereospecific alcohol dehydrogenase isolated from Candida parapsilosis, amino acid and DNA sequences therefor, and method of preparation thereof |
US5798234A (en) * | 1993-10-18 | 1998-08-25 | Degussa Aktiengesellschaft | Method for the directed modification of enzymes, modified enzymes and their use |
US5811238A (en) * | 1994-02-17 | 1998-09-22 | Affymax Technologies N.V. | Methods for generating polynucleotides having desired characteristics by iterative selection and recombination |
US5830721A (en) * | 1994-02-17 | 1998-11-03 | Affymax Technologies N.V. | DNA mutagenesis by random fragmentation and reassembly |
US5837458A (en) * | 1994-02-17 | 1998-11-17 | Maxygen, Inc. | Methods and compositions for cellular and metabolic engineering |
US6117679A (en) * | 1994-02-17 | 2000-09-12 | Maxygen, Inc. | Methods for generating polynucleotides having desired characteristics by iterative selection and recombination |
US5605793A (en) * | 1994-02-17 | 1997-02-25 | Affymax Technologies N.V. | Methods for in vitro recombination |
US5958715A (en) * | 1995-06-26 | 1999-09-28 | Brf International | Method for quantitative measurement of an enzyme linked immunosorbent assay |
US6030779A (en) * | 1995-07-18 | 2000-02-29 | Diversa Corporation | Screening for novel bioactivities |
US5958672A (en) * | 1995-07-18 | 1999-09-28 | Diversa Corporation | Protein activity screening of clones having DNA from uncultivated microorganisms |
US6054267A (en) * | 1995-12-07 | 2000-04-25 | Diversa Corporation | Method for screening for enzyme activity |
US5854035A (en) * | 1996-02-22 | 1998-12-29 | Degussa Ag | Enzyme with leuDH activity, nucleotide sequence coding therefor and process for the prepartion of the enzyme |
US6001574A (en) * | 1996-06-18 | 1999-12-14 | Diversa Corporation | Production and use of normalized DNA libraries |
US5965408A (en) * | 1996-07-09 | 1999-10-12 | Diversa Corporation | Method of DNA reassembly by interrupting synthesis |
US5801006A (en) * | 1997-02-04 | 1998-09-01 | Specialty Assays, Inc. | Use of NADPH and NADH analogs in the measurement of enzyme activities and metabolites |
US20020192786A1 (en) * | 1997-04-23 | 2002-12-19 | Kaneka Corporation | Producing optically active amino compounds |
US6727083B2 (en) * | 1999-03-19 | 2004-04-27 | Sumitomo Chemical Company, Limited | Protein capable of catalyzing transamination stereoselectively, gene encoding said protein and use thereof |
US6365380B2 (en) * | 2000-02-23 | 2002-04-02 | Pcbu Services, Inc. | Method for stereoselectively inverting a chiral center of a chemical compound using an enzyme and a metal catalyst |
US20030138930A1 (en) * | 2000-11-14 | 2003-07-24 | Applera Corporation | Isolated human dehydrogenaseproteins, nucleic acid molecules encoding these human dehydrogenase proteins, and uses thereof |
Cited By (76)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8617864B2 (en) | 2006-10-02 | 2013-12-31 | Codexis, Inc. | Polynucleotides encoding ketoreductases for producing stereoisomerically pure statins and synthetic intermediates therefor |
US20080248539A1 (en) * | 2006-10-02 | 2008-10-09 | Codexis, Inc. | Compositions and methods for producing stereoisomerically pure statins and synthetic intermediates therefor |
US8273547B2 (en) | 2006-10-02 | 2012-09-25 | Codexis, Inc. | Engineered ketoreductases and methods for producing stereoisomerically pure statins |
US7879585B2 (en) | 2006-10-02 | 2011-02-01 | Codexis, Inc. | Ketoreductase enzymes and uses thereof |
US20110165670A1 (en) * | 2007-02-08 | 2011-07-07 | Codexis, Inc. | Ketoreductases and Uses Thereof |
US20080318295A1 (en) * | 2007-02-08 | 2008-12-25 | Codexis, Inc. | Ketoreductases and Uses Thereof |
US8415127B2 (en) | 2007-02-08 | 2013-04-09 | Codexis, Inc. | Ketoreductases and uses thereof |
US8980605B2 (en) | 2007-02-08 | 2015-03-17 | Codexis, Inc. | Ketoreductases and uses thereof |
US7820421B2 (en) | 2007-02-08 | 2010-10-26 | Codexis, Inc. | Ketoreductases and uses thereof |
US8071347B2 (en) | 2007-02-08 | 2011-12-06 | Codexis, Inc. | Ketoreductases and uses thereof |
US8227229B2 (en) | 2007-08-24 | 2012-07-24 | Codexis, Inc. | Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane |
US10851397B2 (en) | 2007-08-24 | 2020-12-01 | Codexis, Inc. | Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane |
US7977078B2 (en) | 2007-08-24 | 2011-07-12 | Codexis, Inc. | Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane |
US20110217754A1 (en) * | 2007-08-24 | 2011-09-08 | Codexis, Inc. | Ketoreductase polypeptides for the production of (r)-3-hydroxythiolane |
US9394552B2 (en) | 2007-08-24 | 2016-07-19 | Codexis, Inc. | Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane |
US8962285B2 (en) | 2007-08-24 | 2015-02-24 | Codexis, Inc. | Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane |
US11427841B2 (en) | 2007-08-24 | 2022-08-30 | Codexis, Inc. | Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane |
US10385371B2 (en) | 2007-08-24 | 2019-08-20 | Codexis, Inc. | Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane |
US20090093031A1 (en) * | 2007-08-24 | 2009-04-09 | Codexis, Inc. | Ketoreductase Polypeptides for the Production of (R)-3-Hydroxythiolane |
US11965194B2 (en) | 2007-08-24 | 2024-04-23 | Codexis, Inc. | Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane |
US9932615B2 (en) | 2007-08-24 | 2018-04-03 | Codexis, Inc. | Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane |
US10100288B2 (en) | 2007-09-13 | 2018-10-16 | Codexis, Inc. | Ketoreductase polypeptides for the reduction of acetophenones |
US9951318B1 (en) | 2007-09-13 | 2018-04-24 | Codexis, Inc. | Ketoreductase polypeptides for the reduction of acetophenones |
US10227572B2 (en) | 2007-09-13 | 2019-03-12 | Codexis, Inc. | Ketoreductase polypeptides for the reduction of acetophenones |
US8512973B2 (en) | 2007-09-13 | 2013-08-20 | Codexis, Inc. | Methods of using engineered ketoreductase polypeptides for the stereoselective reduction of acetophenones |
US11479756B2 (en) | 2007-09-13 | 2022-10-25 | Codexis, Inc. | Ketoreductase polypeptides for the reduction of acetophenones |
US9873863B2 (en) | 2007-09-13 | 2018-01-23 | Codexis, Inc. | Polynucleotides encoding ketoreductase polypeptides for reduction of acetophenones |
US8748143B2 (en) | 2007-09-13 | 2014-06-10 | Codexis, Inc. | Ketoreductase polypeptides for the reduction of acetophenones |
US8852909B2 (en) | 2007-09-13 | 2014-10-07 | Codexis, Inc. | Ketoreductase polypeptides for the reduction of acetophenones |
US9528131B2 (en) | 2007-09-13 | 2016-12-27 | Codexis, Inc. | Methods of using ketoreductase polypeptides for reduction of acetophenones |
US10927351B2 (en) | 2007-09-13 | 2021-02-23 | Codexis, Inc. | Ketoreductase polypeptides for the reduction of acetophenones |
US20090155863A1 (en) * | 2007-09-28 | 2009-06-18 | Codexis, Inc. | Ketoreductase polypeptides and uses thereof |
US8617853B2 (en) | 2007-09-28 | 2013-12-31 | Codexis, Inc. | Ketoreductase polypeptides for the production of (S,E)-methyl 2-(3-(3-(2-(7-chloroquinolin-2-yl)vinyl)phenyl)-3-hydroxypropyl)benzoate |
US8088610B2 (en) | 2007-09-28 | 2012-01-03 | Codexis, Inc. | Ketoreductases for the production of (S,E)-methyl 2-(3-(3-(2-(7-chloroquinolin-2-yl)vinyl)phenyl)-3-hroxypropyl)benzoate |
US10329540B2 (en) | 2007-10-01 | 2019-06-25 | Codexis, Inc. | Ketoreductase polypeptides for the production of azetidinone |
US20110159567A1 (en) * | 2007-10-01 | 2011-06-30 | Codexis, Inc. | Ketoreductase polypeptides for the production of azetidinone |
US20090162909A1 (en) * | 2007-10-01 | 2009-06-25 | Codexis, Inc. | Ketoreductase Polypeptides for the Production of Azetidinone |
US9133442B2 (en) | 2007-10-01 | 2015-09-15 | Codexis, Inc. | Ketoreductase polypeptides for the production of azetidinone |
US11078466B2 (en) | 2007-10-01 | 2021-08-03 | Codexis, Inc. | Ketoreductase polypeptides for the production of azetidinone |
US9382519B2 (en) | 2007-10-01 | 2016-07-05 | Codexis, Inc. | Ketoreductase polypeptides for the production of azetidinone |
US7883879B2 (en) | 2007-10-01 | 2011-02-08 | Codexis, Inc. | Ketoreductase polypeptides for the production of azetidinone |
US8980606B2 (en) | 2007-10-01 | 2015-03-17 | Codexis, Inc. | Ketoreductase polypeptides for the production of azetidinone |
US10597641B2 (en) | 2007-10-01 | 2020-03-24 | Codexis, Inc. | Ketoreductase polypeptides for the production of azetidinone |
US9476034B2 (en) | 2007-10-01 | 2016-10-25 | Codexis, Inc. | Ketoreductase polypeptides for the production of azetidinone |
US8257952B2 (en) | 2007-10-01 | 2012-09-04 | Codexis, Inc. | Ketoreductase polypeptides for the production of azetidinone |
US9580694B2 (en) | 2007-10-01 | 2017-02-28 | Codexis, Inc. | Ketoreductase polypeptides for the production of azetidinone |
US8470572B2 (en) | 2007-10-01 | 2013-06-25 | Codexis, Inc. | Ketoreductase polypeptides for the production of azetidinone |
US9719071B2 (en) | 2007-10-01 | 2017-08-01 | Codexis, Inc. | Ketoreductase polypeptides for the production of azetidinone |
US8288141B2 (en) | 2008-08-27 | 2012-10-16 | Codexis, Inc. | Ketoreductase polypeptides for the production of 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine |
US20100151534A1 (en) * | 2008-08-27 | 2010-06-17 | Codexis, Inc. | Ketoreductase polypeptides for the production of a 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine |
US8673607B2 (en) | 2008-08-27 | 2014-03-18 | Codexis, Inc. | Ketoreductase polypeptides for the production of a 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine |
US20100173369A1 (en) * | 2008-08-27 | 2010-07-08 | Codexis, Inc. | Ketoreductase polypeptides for the production of 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine |
US8426178B2 (en) | 2008-08-27 | 2013-04-23 | Codexis, Inc. | Ketoreductase polypeptides for the production of a 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine |
US10544401B2 (en) | 2008-08-29 | 2020-01-28 | Codexis, Inc. | Ketoreductase polypeptides |
US10988739B2 (en) | 2008-08-29 | 2021-04-27 | Codexis, Inc. | Ketoreductase polypeptides |
US8415126B2 (en) | 2008-08-29 | 2013-04-09 | Codexis, Inc. | Polynucleotides encoding recombinant ketoreductase polypeptides |
US10047348B2 (en) | 2008-08-29 | 2018-08-14 | Codexis, Inc. | Ketoreductase polypeptides |
US10246687B2 (en) | 2008-08-29 | 2019-04-02 | Codexis, Inc. | Ketoreductase polypeptides |
US8273554B2 (en) | 2008-08-29 | 2012-09-25 | Codexis, Inc. | Ketoreductase polypeptides for the stereoselective production of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one |
US9139820B2 (en) | 2008-08-29 | 2015-09-22 | Codexis, Inc. | Ketoreductase polypeptides |
US8956840B2 (en) | 2008-08-29 | 2015-02-17 | Codexis, Inc. | Engineered ketoreductase polypeptides |
US9422530B2 (en) | 2008-08-29 | 2016-08-23 | Codexis, Inc. | Ketoreductase polypeptides |
US9796964B2 (en) | 2008-08-29 | 2017-10-24 | Codexis, Inc. | Ketoreductase polypeptides |
US11345898B2 (en) | 2009-08-19 | 2022-05-31 | Codexis, Inc. | Ketoreductase polypeptides for the preparation of phenylephrine |
US10870835B2 (en) | 2009-08-19 | 2020-12-22 | Codexis, Inc. | Ketoreductase polypeptides for the preparation of phenylephrine |
US10358631B2 (en) | 2009-08-19 | 2019-07-23 | Codexis, Inc. | Ketoreductase polypeptides for the preparation of phenylephrine |
US10590396B2 (en) | 2009-08-19 | 2020-03-17 | Codexis, Inc. | Ketoreductase polypeptides for the preparation of phenylephrine |
US9834758B2 (en) | 2009-08-19 | 2017-12-05 | Codexis, Inc. | Ketoreductase polypeptides for the preparation of phenylephrine |
US9102959B2 (en) | 2009-08-19 | 2015-08-11 | Codexis, Inc. | Ketoreductase polypeptides for the preparation of phenylephrine |
US10544400B2 (en) | 2010-05-04 | 2020-01-28 | Codexis, Inc. | Biocatalysts for ezetimibe synthesis |
US9040262B2 (en) | 2010-05-04 | 2015-05-26 | Codexis, Inc. | Biocatalysts for ezetimibe synthesis |
US9388391B2 (en) | 2010-05-04 | 2016-07-12 | Codexis, Inc. | Biocatalysts for Ezetimibe synthesis |
US10053673B2 (en) | 2010-05-04 | 2018-08-21 | Codexis, Inc. | Biocatalysts for Ezetimibe synthesis |
US10995320B2 (en) | 2010-05-04 | 2021-05-04 | Codexis, Inc. | Biocatalysts for Ezetimibe synthesis |
US9644189B2 (en) | 2010-05-04 | 2017-05-09 | Codexis, Inc. | Biocatalysts for ezetimibe synthesis |
CN112394050A (en) * | 2019-08-19 | 2021-02-23 | 中国科学院天津工业生物技术研究所 | Detection method for high-throughput screening of ketone compounds and application of detection method in enzyme screening |
Also Published As
Publication number | Publication date |
---|---|
US20080076162A1 (en) | 2008-03-27 |
AU2002232603A1 (en) | 2002-05-15 |
US7202070B2 (en) | 2007-04-10 |
US7642073B2 (en) | 2010-01-05 |
WO2002036742A3 (en) | 2003-08-21 |
WO2002036742A2 (en) | 2002-05-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7642073B2 (en) | Method for reductive amination of a ketone using a mutated enzyme | |
Chaparro‐Riggers et al. | Comparison of three enoate reductases and their potential use for biotransformations | |
Resch et al. | Novel carbon–carbon bond formations for biocatalysis | |
Goetz et al. | Continuous production of (R)‐phenylacetylcarbinol in an enzyme‐membrane reactor using a potent mutant of pyruvate decarboxylase from Zymomonas mobilis | |
Lan Tee et al. | Directed evolution of oxygenases: screening systems, success stories and challenges | |
Engel et al. | Acetohydroxyacid synthase: a new enzyme for chiral synthesis of R‐phenylacetylcarbinol | |
Cosgrove et al. | Imine reductases, reductive aminases, and amine oxidases for the synthesis of chiral amines: discovery, characterization, and synthetic applications | |
EP1179595B1 (en) | (r)-2-octanol dehydrogenases, methods for producing the enzymes, dna encoding the enzymes, and methods for producing alcohols using the enzymes | |
Löwe et al. | Enantioselective synthesis of amines via reductive amination with a dehydrogenase mutant from Exigobacterium sibiricum: Substrate scope, co-solvent tolerance and biocatalyst immobilization | |
Fesko | Comparison of L-threonine aldolase variants in the aldol and retro-aldol reactions | |
Barber et al. | Continuous colorimetric screening assay for detection of d-amino acid aminotransferase mutants displaying altered substrate specificity | |
Matsuda et al. | Conversion of pyrrole to pyrrole-2-carboxylate by cells of Bacillus megaterium in supercritical CO2 | |
PT1499716E (en) | Adh from rhodococcus erythropolis | |
Zeng et al. | Integrating enzyme evolution and high-throughput screening for efficient biosynthesis of l-DOPA | |
Walton et al. | A high-throughput assay for screening L-or D-amino acid specific aminotransferase mutant libraries | |
Butler et al. | Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by E. coli resting cells | |
Wu et al. | Efficient enzymatic synthesis of α-keto acids by redesigned substrate-binding pocket of the L-amino acid deaminase (PmiLAAD) | |
Truppo | Rapid screening and process development of biocatalytic reactions | |
Bulut et al. | Development of a growth-dependent selection system for identification of L-threonine aldolases | |
US11441142B2 (en) | FLS variant having increased activity | |
US20230107679A1 (en) | Method For Preparing (S)-1,2,3,4-Tetrahydroisoquinoline-1 Carboxylic Acid and Derivatives Thereof | |
CA2497499A1 (en) | Use of malate dehydrogenase for nadh regeneration | |
Ravot et al. | High throughput discovery of alcohol dehydrogenases for industrial biocatalysis | |
Anderson et al. | Development of a multienzyme reactor for dopamine synthesis: I. Enzymology and kinetics | |
Zhu et al. | High-throughput assay of tyrosine phenol-lyase activity using a cascade of enzymatic reactions |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: BIOCATALYTICS, INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:ROZZELL, J. DAVID, JR.;REEL/FRAME:012465/0030 Effective date: 20011024 |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
AS | Assignment |
Owner name: CODEXIS, INC., CALIFORNIA Free format text: NUNC PRO TUNC ASSIGNMENT;ASSIGNOR:BIOCATALYTICS, INC.;REEL/FRAME:020478/0612 Effective date: 20080205 |
|
FEPP | Fee payment procedure |
Free format text: PAYER NUMBER DE-ASSIGNED (ORIGINAL EVENT CODE: RMPN); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY Free format text: PAYOR NUMBER ASSIGNED (ORIGINAL EVENT CODE: ASPN); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
FPAY | Fee payment |
Year of fee payment: 4 |
|
FPAY | Fee payment |
Year of fee payment: 8 |
|
MAFP | Maintenance fee payment |
Free format text: PAYMENT OF MAINTENANCE FEE, 12TH YR, SMALL ENTITY (ORIGINAL EVENT CODE: M2553); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY Year of fee payment: 12 |